Detection Of Bacteria Belonging to the Genus Campylobacter By Targeting Cytolethal Distending Toxin

ABSTRACT

Multiplex PCR primers that can amplify the cdt genes of  C. jejuni, C. coli , and  C. fetus  in a bacterial species-specific manner were prepared. Multiplex PCR with the primers was assessed using  Campylobacter  bacteria, other cdt gene-positive bacteria, and representative bacteria responsible for enteric infection. As a result, the present inventors&#39; multiplex PCR using cdtB amplification primers was proven to enable simultaneous detection of different  Campylobacter  bacteria with high specificity. The methods of the present invention can identify  Campylobacter  at the bacterial species level in a single manipulation even when domestic animals or humans are infected with different bacterial species of  Campylobacter.

FIELD OF THE INVENTION

The present invention relates to methods of detecting Campylobacterbacteria in a sample by targeting the cytolethal distending toxin ofCampylobacter bacteria.

BACKGROUND OF THE INVENTION

Cultivation test is commonly used to identify bacterial species ofCampylobacter bacteria. However, the test requires complex andsubstantial effort, because some bacterial species are difficult toidentify based on their biochemical properties alone. Also, the bacteriaare microaerophilic and some bacterial species should be cultured atdifferent temperatures. The cultivation test for Campylobacter bacteriausually takes a long time, seven to ten days, including isolation andidentification.

To date, Campylobacter jejuni (hereinafter abbreviated as “C. jejuni”)and Campylobacter coli (hereinafter abbreviated as “C. coli”) accountfor about 94% and 4% of Campylobacter bacteria isolated from diarrheapatients, respectively. Thus, the two bacterial species comprise themajority of Campylobacter bacteria. Accordingly, in most cases, test forCampylobacter bacteria in clinical practice only covers C. jejuni and C.coli which are specified as food poisoning bacteria. Furthermore,selection media commonly used in the test are those developed for mainlyC. jejuni and C. coli, and in general, the culture is carried out at 42°C. Therefore, it is hard to say that the test covers Campylobacter fetus(hereinafter abbreviated as “C. fetus”) which has differenttemperature-sensitive property from C. jejuni and C. coli or otherCampylobacter bacteria. Meanwhile, a mass outbreak of food poisoningcaused by C. fetus occurred in Osaka in 2005. Infection with C. fetuscauses not only gastroenteritis such as diarrhea but also other severesymptoms such as sepsis and meningitis in human. Furthermore, infectionwith C. fetus can result in infertility, miscarriage, or the like inanimals such as cattle. It is thus important to improve the test systemfor Campylobacter bacteria including C. fetus.

It is difficult to rapidly identify bacterial species of Campylobacterbacteria based on their biochemical properties, and some ofCampylobacter species often cannot be distinguished based on theirbiochemical properties because of their close resemblance. Inparticular, C. jejuni and C. coli are problematic because they aredistinguished based on the presence of hippuricase activity, and whenthe enzyme activity is low, C. jejuni is falsely identified as C. coli.For this reason, PCR methods for detecting the presence of thehippuricase gene have been used in actual tests. In recent years, 16SrRNA gene analysis is frequently used as a method for identifyingbacterial species at the gene level. However, C. jejuni and C. coli arehighly homologous to each other, and thus often cannot be distinguishedfrom each other by the 16S rRNA gene analysis.

To solve the above-described problems, the present inventors focused andconducted academic research on cytolethal distending toxin (CDT) ofCampylobacter bacteria (Asakura M. et al., Microbial Pathogenesis 42(2007) 174-183; Yamasaki S. et al., Toxin Reviews, 25: 61-88, 2006), anddeveloped a method for detecting Campylobacter bacteria using thecytolethal distending toxin genes (cdtA, cdtB, and cdtC) (WO2005/054472). However, there is an increasing trend in both theCampylobacter infection rate and the number of patients, and thusdevelopment of simpler and more rapid methods for identifyingCampylobacter bacteria is much expected (“Food poisoning outbreak foreach causative agent”, the Ministry of Health, Labor and Welfare ofJapan).

SUMMARY OF THE INVENTION

The present invention was achieved in view of the circumstancesdescribed above. An objective of the present invention is to providenovel methods for detecting Campylobacter bacteria using their cdtgenes.

The present inventors conducted dedicated studies to achieve the aboveobjective. The present inventors prepared multiplex PCR primers capableof amplifying the cdt genes of C. jejuni, C. coli, and C. fetus in abacterial species-specific manner. Multiplex PCR was assessed usingCampylobacter bacteria including many clinical isolates, other cdtgene-positive bacteria, and representative bacteria that cause entericinfection. The present inventors also aimed to simultaneously detectmultiple bacterial species of Campylobacter by multiplex PCR using cdtBamplification primers. The result demonstrated that the presentinventors' multiplex PCR method using cdtB amplification primers wascapable of simultaneously detecting multiple bacterial species ofCampylobacter in a highly specific manner. Even when domestic animals orhumans have mixed infection with multiple bacterial species ofCampylobacter, the method of the present invention enablesidentification of Campylobacter bacteria at the bacterial species levelin a single manipulation. Specifically, the present invention relates tomethods for detecting Campylobacter bacteria by amplifying the cdt genesof Campylobacter bacteria, and more specifically provides the following:

[1] a method for detecting a Campylobacter bacterium in a test sample,which comprises the step of nucleic acid amplification reaction in thetest sample using one or more of primer pairs that comprise twopolynucleotides that can specifically bind to genomic DNA or mRNA ofcdtB of the Campylobacter bacterium, wherein the primer pairs are:

(a) a primer pair capable of amplifying a genomic DNA region of cdtB ofthe Campylobacter bacterium which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 1 and 2, or an mRNA regioncorresponding to the amplifiable genomic DNA region; and

(b) a primer pair capable of amplifying a genomic DNA region of cdtB ofthe Campylobacter bacterium which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 3 and 4, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

-   [2] the method of [1], wherein the nucleic acid amplification    reaction is carried out using primer pairs (a) and (b); and

(c) a primer pair capable of amplifying a genomic DNA region of cdtB ofthe Campylobacter bacterium which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 5 and 6, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

[3] the method of [1] or [2], which comprises, before or after the stepof nucleic acid amplification reaction, an additional nucleic acidamplification reaction step using a common primer pair comprising twopolynucleotides that can commonly bind to the genomic DNA or mRNA of anyone of cdtA, cdtB, and cdtC of Campylobacter bacteria;

[4] the method of [3], wherein the common primer pair is any one of: aprimer pair comprising the sequences of SEQ ID NOs: 7 and 8; a primerpair comprising the sequences of SEQ ID NOs: 9 and 10; a primer paircomprising a combination of two sequences selected from the foursequences of SEQ ID NOs: 11, 12, 13, and 14; a primer pair comprisingthe sequences of SEQ ID NOs: 15 and 16; and a primer pair comprising thesequences of SEQ ID NOs: 17 and 18;

[5] a kit for use in the method of [1], which comprises a manual and atleast either:

(a) a primer pair capable of amplifying a genomic DNA region of cdtB ofthe Campylobacter bacterium which is amplified with the primer paircomprising the sequences of SEQ ID NOs: 1 and 2, or an mRNA regioncorresponding to the amplifiable genomic DNA region; or

(b) a primer pair capable of amplifying a genomic DNA region of cdtB ofthe Campylobacter bacterium which is amplified with the primer paircomprising the sequences of SEQ ID NOs: 3 and 4, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

each of which comprises two polynucleotides that can specifically bindto the genomic DNA or mRNA of cdtB of the Campylobacter bacterium;

[6] the kit of [5], which further comprises:

(c) a primer pair capable of amplifying a genomic DNA region of cdtB ofthe Campylobacter bacterium which is amplified with the primer paircomprising the sequences of SEQ ID NOs: 5 and 6, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

[7] a method for detecting a Campylobacter bacterium in a test sample,which comprises the step of nucleic acid amplification reaction in thetest sample using a primer pair comprising two polynucleotides that canspecifically bind to genomic DNA or mRNA of cdtA of a Campylobacterbacterium, wherein the primer pair is:

(a) a primer pair capable of amplifying a genomic DNA region of cdtA ofthe Campylobacter bacterium which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 19 and 20, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

[8] The method of [7], wherein the nucleic acid amplification reactionis carried out using primer pair (a), and

(b) a primer pair capable of amplifying a genomic DNA region of cdtA ofthe Campylobacter bacterium which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 21 and 22, or an mRNA regioncorresponding to the amplifiable genomic DNA region; and

(c) a primer pair capable of amplifying a genomic DNA region of cdtA ofthe Campylobacter bacterium which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 23 and 24, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

[9] a kit for use in the method of [7], which comprises a manual and aprimer pair comprising two polynucleotides that can specifically bind tothe genomic DNA or mRNA of cdtA of the Campylobacter bacterium, whereinthe primer pair is:

(a) a primer pair capable of amplifying a genomic DNA region of cdtA ofthe Campylobacter bacterium which is amplified with the primer paircomprising the sequences of SEQ ID NOs: 19 and 20, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

[10] the kit of [9], which further comprises:

(b) a primer pair capable of amplifying a genomic DNA region of cdtA ofthe Campylobacter bacterium which is amplified with the primer paircomprising the sequences of SEQ ID NOs: 21 and 22, or an mRNA regioncorresponding to the amplifiable genomic DNA region; and

(c) a primer pair capable of amplifying a genomic DNA region of cdtA ofthe Campylobacter bacterium which is amplified with the primer paircomprising the sequences of SEQ ID NOs: 23 and 24, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

-   [11] a method for detecting a Campylobacter bacterium in a test    sample, which comprises the step of nucleic acid amplification    reaction in the test sample using a primer pair comprising two    polynucleotides that can specifically bind to genomic DNA or mRNA of    cdtC of a Campylobacter bacterium, wherein the primer pair is:

(a) a primer pair capable of amplifying a genomic DNA region of cdtC ofthe Campylobacter bacterium which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 25 and 26, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

-   [12] the method of [11], wherein the nucleic acid amplification    reaction is carried out using primer pair (a), and

(b) a primer pair capable of amplifying a genomic DNA region of cdtC ofthe Campylobacter bacterium which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 27 and 28, or an mRNA regioncorresponding to the amplifiable genomic DNA region; and

(c) a primer pair capable of amplifying a genomic DNA region of cdtC ofthe Campylobacter bacterium which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 29 and 30, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

[13] a kit for use in the method of [11], which comprises a manual and aprimer pair comprising two polynucleotides that can specifically bind tothe genomic DNA or mRNA of cdtC of the Campylobacter bacterium, whereinthe primer pair is:

(a) a primer pair capable of amplifying a genomic DNA region of cdtC ofthe Campylobacter bacterium which is amplified with the primer paircomprising the sequences of SEQ ID NOs: 25 and 26, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

[14] the kit of [13], which further comprises:

(b) a primer pair capable of amplifying a genomic DNA region of cdtC ofthe Campylobacter bacterium which is amplified with the primer paircomprising the sequences of SEQ ID NOs: 27 and 28, or an mRNA regioncorresponding to the amplifiable genomic DNA region; and

(c) a primer pair capable of amplifying a genomic DNA region of cdtC ofthe Campylobacter bacterium which is amplified with the primer paircomprising the sequences of SEQ ID NOs: 29 and 30, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

[15] a method for detecting a Campylobacter bacterium in a test sample,which comprises the step of nucleic acid amplification reaction in atest sample using one or more of:

(a) a primer pair capable of amplifying a genomic DNA region of cdtC ofa Campylobacter bacterium that is amplified by a primer pair comprisingthe sequences of SEQ ID NOs: 37 and 38, or an mRNA region correspondingto the amplifiable genomic DNA region;

(b) a primer pair capable of amplifying a genomic DNA region of cdtC ofa Campylobacter bacterium that is amplified by a primer pair comprisingthe sequences of SEQ ID NOs: 40 and 41, or an mRNA region correspondingto the amplifiable genomic DNA region; and

(c) a primer pair capable of amplifying a genomic DNA region of cdtC ofa Campylobacter bacterium that is amplified by a primer pair comprisingthe sequences of SEQ ID NOs: 43 and 44, or an mRNA region correspondingto the amplifiable genomic DNA region;

each of which comprises two polynucleotides that can specifically bindto a genomic DNA or mRNA of cdt of the Campylobacter bacterium;

[16] the method of [15], wherein the nucleic acid amplification reactionis achieved by using a quantitative PCR method or quantitative real-timePCR method;

[17] the method of [15] or [16], which further comprises any one or moreof:

(i) the step of detecting the nucleic acid fragment amplified with aprimer pair comprising the sequences of SEQ ID NOs: 37 and 38 by usingthe probe of SEQ ID NO: 39;

(ii) the step of detecting the nucleic acid fragment amplified with aprimer pair comprising the sequences of SEQ ID NOs: 40 and 41 by usingthe probe of SEQ ID NO: 42; and

(iii) the step of detecting the nucleic acid fragment amplified with aprimer pair comprising the sequences of SEQ ID NOs: 43 and 44 by usingthe probe of SEQ ID NO: 45;

[18] a kit for use in the method of [15], which comprises a manual andat least one of:

(a) a primer pair capable of amplifying a genomic DNA region of cdt of aCampylobacter bacterium that is amplified by the primer pair comprisingthe sequences of SEQ ID NOs: 37 and 38, or an mRNA region correspondingto the amplifiable genomic DNA region;

(b) a primer pair capable of amplifying a genomic DNA region of cdt of aCampylobacter bacterium that is amplified by the primer pair comprisingthe sequences of SEQ ID NOs: 40 and 41, or an mRNA region correspondingto the amplifiable genomic DNA region; and

(c) a primer pair capable of amplifying a genomic DNA region of cdt of aCampylobacter bacterium that is amplified by the primer pair comprisingthe sequences of SEQ ID NOs: 43 and 44, or an mRNA region correspondingto the amplifiable genomic DNA region;

each of which comprises two polynucleotides that can specifically bindto a genomic DNA or mRNA of cdt of the Campylobacter bacterium;

[19] the kit of [18], which further comprises at least one of the probesof SEQ ID NOs: 39, 42, and 45.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents photographs showing the result of multiplex PCRtargeting the cdtA, cdtB, and cdtC genes. Multiplex PCR was carried outusing a boiled template of each bacterial strain, and the PCR productswere analyzed by 2% agarose gel electrophoresis: (a) cdtA gene; (b) cdtBgene; (c) cdtC gene. The PCR products and molecular weight marker wereloaded at 5 μl per lane.

Lanes 1 and 16, 100-bp Ladder marker; lane 2, C. jejuni ATCC33560; lane3, C. jejuni ATCC43432; lane 4, C. coli ATCC33559; lane 5, C. coliATCC43478; lane 6, C. fetus ATCC27374; lane 7, C. fetus ATCC19438; lane8, C. hyointestinalis ATCC35217; lane 9, C. lari ATCC43675; lane 10, C.upsaliensis ATCC43954; lane 11, C. helveticus ATCC51209; lane 12, H.hepaticus ATCC51449; lane 13, Haemophilus ducreyi (H. ducreyi)ATCC700724; lane 14, A. actinomycetemcomitans S01; lane 15, E. coliC600.

FIG. 2 presents a photograph showing the result of PCR using commonprimers targeting the cdtB gene of various Campylobacter bacteria. PCRwas carried out with common primers targeting the cdtB gene using aboiled template for each bacterial strain. The PCR products wereanalyzed by 2% agarose gel electrophoresis. The PCR products andmolecular weight marker were loaded at 5 ml per lane.

FIG. 3 presents a photograph showing the result of multiplex PCRtargeting the cdtB gene using template DNAs from several bacterialspecies. Boiled templates of C. jejuni Col-008, C. coli Col-192, and C.fetus Col-187 were mixed in various combinations (1 μl each). MultiplexPCR targeting the cdtB gene was carried out using the mixtures. The PCRproducts were analyzed by 2% agarose gel electrophoresis. The PCRproducts and molecular weight marker were loaded at 5 μl per lane.

FIG. 4 presents a photograph showing the detection limit of multiplexPCR targeting the cdtB gene. Multiplex PCR targeting the cdtB gene wascarried out using a boiled template of each bacterial strain so that ineach PCR tube 10° colony forming units (cfu) become 10³ cfu. PCRproducts were analyzed by 2% agarose gel electrophoresis. The PCRproducts and molecular weight marker were loaded at 5 ml per lane.

FIG. 5-1 is a diagram showing the binding sites of primer comprising thesequence of SEQ ID NO: 1 (primer name: Cj-CdtBU5) and primer comprisingthe sequence of SEQ ID NO: 2 (primer name: Cj-CdtBR6) within the genomicDNA sequence of cdtB of C. jejuni 81-176. The binding site for eachprimer is underlined. Asterisk (*) indicates the stop codon. The aminoacid sequences shown below the nucleotide sequence are those of CDTsubunits encoded by in the order of cdtA, cdtB, and cdtC.

FIG. 5-2 is the continuation of FIG. 5-1.

FIG. 5-3 is the continuation of FIG. 5-2.

FIG. 6-1 presents a diagram showing the binding sites of primercomprising the sequence of SEQ ID NO: 3 (primer name: Cf-CdtBU6) andprimer comprising the sequence of SEQ ID NO: 4 (primer name: Cf-CdtBR3)within the genomic DNA sequence of cdtB of C. fetus Col-187. The bindingsite for each primer is underlined and the primer name is shown. Theunderline that does not have a primer name indicates SD sequence.Asterisk (*) indicates the stop codon. The amino acid sequences shownbelow the nucleotide sequence are those of CDT subunits encoded by inthe order of cdtA, cdtB, and cdtC. Arrow indicates the direction oftranslation for the encoded polypeptides.

FIG. 6-2 is the continuation of FIG. 6-1.

FIG. 7-1 is a diagram showing the binding sites of primer comprising thesequence of SEQ ID NO: 5 (primer name: (Cc-CdtBU5) and primer comprisingthe sequence of SEQ ID NO: 6 (primer name: Cc-CdtBR5) within the genomicDNA sequence of cdtB of C. coli Col-243. The binding site for eachprimer is underlined and the primer name is shown. The underline thatdoes not have a primer name indicates SD sequence. Asterisk (*)indicates the stop codon. The amino acid sequences shown below thenucleotide sequence are those of CDT subunits encoded by in the order ofcdtA, cdtB, and cdtC. Arrow indicates the direction of translation forthe encoded polypeptides.

FIG. 7-2 is the continuation of FIG. 7-1.

FIG. 8 is a graph showing a real-time PCR standard curve for C. jejuni.

FIG. 9 is a graph showing a real-time PCR standard curve for C. coli.

FIG. 10 is a graph showing a real-time PCR standard curve for C. fetus.

FIG. 11-1 is a diagram showing sequence comparison of the cdt genebetween the C. jejuni 81-176 strain (SEQ ID NO: 31) and a cdtgene-deficient C. jejuni strain (Genbank Accession No. AY442600).Dot-and-dash line (-•-) indicates the cdtA gene region; solid lineindicates the cdtB gene region; and dotted line indicates the cdtC generegion. (1) The cdtC 3′ end region in the mutant deficient cdt gene(AY442300) remains unknown because there is no report on the 3′ end. Dot“.” represents the same nucleotide as that of the 81-176 strain and bar“-” represents gap.

FIG. 11-2 is the continuation of FIG. 11-1.

FIG. 12-1 is a diagram showing comparison the cdtC gene between C.jejuni and a gene-deficient mutant strain of C. jejuni (GenbankAccession No. AY442300), and positions of the real-time PCR primers andprobe.

FIG. 12-2 is the continuation of FIG. 12-1.

FIG. 12-3 is the continuation of FIG. 12-2.

FIG. 12-4 is the continuation of FIG. 12-3.

FIG. 13-1 is a diagram showing comparison of the cdt gene between the C.coli

Col-243 strain (SEQ ID NO: 33) and other C. coli strains, and positionsof the real-time PCR primers and probe. Dot-and-dash line (-•-)indicates the cdtA gene region; solid line indicates the cdtB generegion; and dotted line indicates the cdtC gene region. Dot “.”represents the same nucleotide as that of the Col-243 strain and bar “-”represents gap.

FIG. 13-2 is the continuation of FIG. 13-1.

FIG. 13-3 is the continuation of FIG. 13-2.

FIG. 13-4 is the continuation of FIG. 13-3.

FIG. 13-5 is the continuation of FIG. 13-4.

FIG. 13-6 is the continuation of FIG. 13-5.

FIG. 13-7 is the continuation of FIG. 13-6.

FIG. 13-8 is the continuation of FIG. 13-7.

FIG. 13-9 is the continuation of FIG. 13-8.

FIG. 13-10 is the continuation of FIG. 13-9.

FIG. 13-11 is the continuation of FIG. 13-10.

FIG. 13-12 is the continuation of FIG. 13-11.

FIG. 13-13 is the continuation of FIG. 13-12.

FIG. 13-14 is the continuation of FIG. 13-13.

FIG. 14-1 is a diagram showing sequence comparison of the cdt genebetween the C. fetus Col-187 strain (SEQ ID NO: 32) and C. fetus C90strain derived from Thailand. Dot-and-dash line (-•-) indicates the cdtAgene region; solid line indicates the cdtB gene region; and dotted lineindicates the cdtC gene region. Dot “.” represents the same nucleotideas that of the Col-187cdt strain and bar “-” represents gap.

FIG. 14-2 is the continuation of FIG. 14-1.

FIG. 14-3 is the continuation of FIG. 14-2.

FIG. 15-1 is a diagram showing comparison of the cdtC gene between theC. fetus and C. fetus C90 strain derived from Thailand, and positions ofthe real-time PCR primers and probe.

FIG. 15-2 is the continuation of FIG. 15-1.

FIG. 15-3 is the continuation of FIG. 15-2.

FIG. 15-4 is the continuation of FIG. 15-3.

DETAILED DESCRIPTION OF THE INVENTION

Herein, the phrase “cytolethal distending toxins” (CDTs or CLDTs) refersto toxic factors belonging to the group of proteinaceous type A-Bholotoxins. The cytolethal distending toxin has a subunit structureconsisting of three subunits A, B, and C. It is believed that subunit Bis the active site unit of the toxin and subunits A and B are involvedin cell adhesion. When the toxin acts on cells, it causes celldeformation such as cell swelling, and finally leads to cell death. Celldeformation such as cell swelling is also observed when heat-labileenterotoxin (LT), which is produced by toxigenic E. coli, or the like isexperimentally allowed to act on cells. When the toxin is inactivated,however, the cells recover and survive. In contrast, cells do notrecover but instead are killed, even when CDT is inactivated.

The term “polynucleotide” as used herein refers to a polymer made up ofa number of bases or base pairs consisting of ribonucleotides ordeoxyribonucleotides. Polynucleotides include RNAs, single-stranded DNAsas well as double-stranded DNAs. Polynucleotides herein may include bothunmodified, naturally-occurring polynucleotides and modifiedpolynucleotides. Tritylated bases and special bases, such as inosine,are examples of modified bases.

The term “polypeptide” as used herein refers to a polymer made up of anumber of amino acids. Therefore, oligopeptides and proteins are alsoincluded within the concept of polypeptides. Polypeptides include bothunmodified, naturally-occurring polypeptides and modified polypeptides.Examples of polypeptide modifications include acetylation; acylation;ADP-ribosylation; amidation; covalent binding with flavin; covalentbinding with heme moieties; covalent binding with nucleotides ornucleotide derivatives; covalent binding with lipids or lipidderivatives; covalent binding with phosphatidylinositols; cross-linkage;cyclization; disulfide bond formation; demethylation; covalent crosslinkage formation; cystine formation pyroglutamate formation;formylation; g-carboxylation; glycosylation; GPI-anchor formation;hydroxylation; iodination; methylation; myristoylation; oxidation;proteolytic treatment; phosphorylation; prenylation; racemization;selenoylation; sulfation; transfer RNA-mediated amino acid addition to aprotein such as arginylation; ubiquitination; and the like.

The term “mutation” as used herein refers to changes to the amino acidsof an amino acid sequence, or changes to the bases in a nucleotidesequence (that is, substitution, deletion, addition, or insertion of oneor more amino acids or nucleotides). Therefore, the term “mutant” asused herein refers to amino acid sequences wherein one or more aminoacids are changed, or nucleotide sequences wherein one or morenucleotides are changed. Nucleotide sequence changes in the mutant maychange the amino acid sequence of the polypeptide encoded by thestandard polynucleotide, or not. The mutant may be one that exists innature, such as an allelic mutant, or one not yet identified in nature.The mutant may be conservatively altered, wherein substituted aminoacids retain structural or chemical characteristics similar to those ofthe original amino acid. Rarely, mutants may be substitutednon-conservatively. Computer programs known in the art, such as DNA STARsoftware, can be used to decide which or how many amino acid residues tosubstitute, insert, or delete without inhibiting biological orimmunological activities.

“Deletion” is a change to either an amino acid sequence or nucleotidesequence, wherein one or more amino acid residues or nucleotide residuesare missing as compared with the amino acid sequence of a naturallyoccurring cytolethal distending toxin polypeptide, or a nucleotidesequence encoding the same.

“Insertion” or “addition” is a change to either an amino acid sequenceor nucleotide sequence, wherein one or more amino acid residues ornucleotide residues are added as compared with the amino acid sequenceof a naturally-occurring cytolethal distending toxin polypeptide, or anucleotide sequence encoding the same.

“Substitution” is a change to either an amino acid sequence ornucleotide sequence, wherein one or more amino acid residues ornucleotide residues are changed to different amino acid residues ornucleotide residues, as compared to the amino acid sequence of anaturally-occurring cytolethal distending toxin polypeptide, or anucleotide sequence encoding the same.

The term “hybridize” as used herein refers to a process wherein anucleic acid chain binds to its complementary chain through theformation of base pairs.

Herein, the term “detection” means both qualitative and quantitativemeasurements. “Quantitation” also refers to semiquantitativemeasurement.

<Detection of the Presence of Campylobacter Bacteria in Test Samples>

The present invention provides methods for detecting Campylobacterbacteria in test samples. Detection of the presence of Campylobacterbacteria in test samples is useful for various purposes such asdiagnosis of Campylobacter infection, rapid test of food contaminatedwith Campylobacter bacteria, validation in each step of food processing,and identification of bacteria responsible for food poisoning outbreak.

The first embodiment of the detection methods of the present inventionincludes methods for detecting Campylobacter bacteria in a test sample,which comprise the step of carrying out the reaction of amplifyingnucleic acids in the test sample using either or both of:

“(a) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdtB which is amplified by a primer paircomprising the sequences of SEQ ID NOs: 1 and 2, or an mRNA regioncorresponding to the amplifiable genomic DNA region” and

“(b) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdtB which is amplified by a primer paircomprising the sequences of SEQ ID NOs: 3 and 4, or an mRNA regioncorresponding to the amplifiable genomic DNA region”,

both of which comprise two polynucleotides that can specifically bind togenomic DNA or mRNA of Campylobacter bacterial cdtB.

The above-described methods are methods that detect bacteria byamplifying a region specific to genomic DNA or mRNA of cdtB for C.jejuni or C. fetus. As for C. jejuni, the primers used in theabove-described methods representatively include the “primer paircomprising the sequences of SEQ ID NOs: 1 and 2 (primers used in theExamples herein: Cj-CdtBU5 and Cj-CdtBR6)”, but are not limited to thesequences. Any other sequences can be used as long as the primer pairscomprise two polynucleotides that can specifically bind to genomic DNAor mRNA of C. jejuni cdtB, and can amplify a region that is amplifiedfrom genomic DNA of C. jejuni cdtB as a template using a “primer paircomprising the sequences of SEQ ID NOs: 1 and 2”, or the correspondingmRNA region. Herein, “specifically bind” means excluding non-specificbinding from the “binding”. FIG. 5 shows the site where the “primer paircomprising the sequences of SEQ ID NOs: 1 and 2” binds to genomic DNA ofcdtB in the C. jejuni 81-176 strain (SEQ ID NO: 31). In addition, a714-bp amplified product is obtained by amplifying genomic DNA of the C.jejuni ATCC33560 strain (DDBJ Accession No. AB274783) or C. jejuniATCC43432 strain (DDBJ Accession No. AB274784) using the primer paircomprising the sequences of SEQ ID NOs: 1 and 2.

Alternatively, as for C. fetus, the primers representatively include the“primer pair comprising the sequences of SEQ ID NOs: 3 and 4 (primersused in the Examples herein: Cf-CdtBU6 and Cf-CdtBR3)”, but are notlimited to the sequences. Any other sequences can be used as long as theprimer pairs comprise two polynucleotides that can specifically bind togenomic DNA or mRNA of C. fetus cdtB, and can amplify a region that isamplified from genomic DNA of C. fetus cdtB as a template using the“primer pair comprising the sequences of SEQ ID NOs: 3 and 4”, or thecorresponding mRNA region. FIG. 6 shows the binding site where the“primer pair comprising the sequences of SEQ ID NOs: 3 and 4” binds togenomic DNA of cdtB in the C. fetus Col-187 strain (SEQ ID NO: 32).Meanwhile, a 553-bp amplified product is obtained by amplifying genomicDNA of the C. fetus ATCC27374 strain (DDBJ Accession No. AB274802) orATCC19438 strain (DDBJ Accession No. AB274803) using the primer paircomprising the sequences of SEQ ID NOs: 3 and 4.

In the methods of the present invention, a single round of nucleic acidamplification reaction can be performed using the following primer pairsindividually or in combination:

“(a) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdtB which is amplified by the primer paircomprising the sequences of SEQ ID NOs: 1 and 2, or an mRNA regioncorresponding to the amplifiable genomic DNA region” and “(b) a primerpair capable of amplifying a genomic DNA region of Campylobacterbacterial cdtB which is amplified by the primer pair comprising thesequences of SEQ ID NOs: 3 and 4, or an mRNA region corresponding to theamplifiable genomic DNA region”. The PCR method in which a number of PCRprimers are used in a single reaction such as in the Examples herein iscalled “multiplex PCR”. Thus, different bacterial species can beidentified by electrophoresing the PCR products and determining the bandsize. The present invention provides methods for detecting Campylobacterbacteria based on nucleic acid amplification methods, representativelyincluding the above-described multiplex PCR, using primers andcombinations thereof which are preferably used for amplifying differentnucleic acid regions. In the present invention, there is no limitationon the type of nucleic acid amplification method, as long as it yieldsamplification products of interest. The PCR method is a specific exampleof nucleic acid amplification method preferably used in the presentinvention. The methods of the present invention may be conducted as aquantitation method by using a real-time PCR method or such.

In the methods of the present invention, a single round of nucleic acidamplification reaction can be performed using “(c) a primer pair capableof amplifying a genomic DNA region of Campylobacter bacterial cdtB whichis amplified with a primer pair comprising the sequences of SEQ ID NOs:5 and 6, or an mRNA region corresponding to the amplifiable genomic DNAregion” (which are primers capable of amplifying a specific region ofgenomic DNA of C. coli cdtB), in combination with the above-described“(a) primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdtB which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 1 and 2, or an mRNA regioncorresponding to the amplifiable genomic DNA region” and/or theabove-described “(b) primer pair capable of amplifying a genomic DNAregion of Campylobacter bacterial cdtB which is amplified with a primerpair comprising the sequences of SEQ ID NOs: 3 and 4, or an mRNA regioncorresponding to the amplifiable genomic DNA region”. Specifically, thepresent invention provides methods that can simultaneously detect thethree bacterial species of Campylobacter, C. jejuni, C. fetus, and C.coli in a test sample. The present inventors demonstrated that the threebacterial species of Campylobacter, C. jejuni, C. fetus, and C. colicould be detected at a time by nucleic acid amplification reaction thatsimultaneously uses the above-described three types of primers. Asdemonstrated in the Examples herein, the methods of the presentinvention have very high specificity because they achieve the detectionof Campylobacter bacteria of interest without erroneous detection ofother bacterial species of Campylobacter. FIG. 7 shows the site wherethe “primer pair comprising the sequences of SEQ ID NOs: 5 and 6(primers used in the Examples herein: Cc-CdtBU5 and Cc-CdtBR5)” binds togenomic DNA of cdtB in the C. coli Col-243 strain (SEQ ID NO: 33).

The methods of the present invention comprise subsequent to theabove-described step of nucleic acid amplification reaction usingprimers specific to C. jejuni, C. fetus, or C. coli, the step of“detecting the presence of Campylobacter bacteria based on the presenceor molecular weight of fragments amplified from the genomic DNA or mRNAof the Campylobacter bacterial cdtB” or the step of “quantifying thefragments amplified from genomic DNA or mRNA of the Campylobacterbacterial cdtB”.

Alternatively, the methods of the present invention may comprise the“step of nucleic acid amplification reaction using a common primer paircomprising two polynucleotides that can commonly bind to the genomic DNAor mRNA of any one of cdtA, cdtB, and cdtC of Campylobacter bacteria”before or after the step of nucleic acid amplification reaction usingprimers specific to C. jejuni, C. fetus, or C. coli. The above-described“common primer pair comprising two polynucleotides that can commonlybind to the genomic DNA or mRNA of any one of cdtA, cdtB, and cdtC ofCampylobacter bacteria” refers to a primer pair capable of amplifyingthe genomic DNA or mRNA encoding any one of cdtA, cdtB, and cdtC in allof the bacterial species C. jejuni, C. fetus, and C. coli. Specificexamples of such primers include the primer pair used in the Examplesherein, which comprises the sequences of SEQ ID NOs: 7 and 8 (commoncdtB primer pair used in the Examples herein: C-CdtBcom1 andC-CdtBcom2). The common primer pairs that are preferably used includenot only the above-described primer pair but also the primer paircomprising the sequences of SEQ ID NOs: 9 (SEQ ID NO: 7 in WO2005/054472) and 10 (SEQ ID NO: 8 in WO 2005/054472) (common cdtB primerpair), primer pairs comprising a combination of two of the foursequences of SEQ ID NOs: 11 (SEQ ID NO: 47 in WO 2005/054472), 12 (SEQID NO: 48 in WO 2005/054472), 13 (SEQ ID NO: 49 in WO 2005/054472), and14 (SEQ ID NO: 50 in WO 2005/054472) (common cdtB primer pairs), primerpair comprising the sequences of SEQ ID NOs: 15 (SEQ ID NO: 64 in WO2005/054472) and 16 (SEQ ID NO: 65 in WO 2005/054472) (common cdtAprimer pair), and primer pair comprising the sequences of SEQ ID NOs: 17(SEQ ID NO: 66 in WO 2005/054472) and 18 (SEQ ID NO: 67 in WO2005/054472) (common cdtC primer pair). WO 2005/054472 describes indetail the fact that the above-described common primer pairs werecapable of amplifying genomic DNA encoding any one of cdtA, cdtB, andcdtC in all of the bacteria C. jejuni, C. fetus, and C. coli. Theimproved sensitivity for detection of Campylobacter bacteria can beexpected when the nucleic acid amplification reaction using primersspecific to C. jejuni, C. fetus, C. coli is combined with the nucleicacid amplification reaction using the above-described “common primerpairs that comprise two polynucleotides capable of commonly binding togenomic DNAs or mRNAs of cdtA, cdtB or cdtC of Campylobacter bacteria”.As described above, the above primer pairs are common primer pairscapable of commonly amplifying genomic DNAs or mRNAs encoding thecytolethal distending toxin of at least the three bacterial species: C.coli, C. jejuni, and C. fetus. The above-described common primer pairsare expected to be capable of amplifying genomic DNAs or mRNAs encodingthe cytolethal distending toxin of not only the above-described threeCampylobacter bacteria but also other Campylobacter bacteria. Likewise,other primer pairs that can amplify the same genomic DNA regions orcorresponding mRNA regions as those amplified with those primer pairsare assumed to be capable of commonly amplifying the genomic regions orcorresponding mRNA regions of the three bacterial species describedabove and other Campylobacter bacteria.

The second embodiment of the methods of the present invention includesmethods for detecting Campylobacter bacteria in a test sample, whichcomprise the step of nucleic acid amplification reaction in the testsample using “(a) a primer pair capable of amplifying a genomic DNAregion of Campylobacter bacterial cdtA, which is amplified with a primerpair comprising the sequences of SEQ ID NOs: 19 and 20, or mRNA regioncorresponding to the amplifiable genomic DNA region”, which comprisestwo polynucleotides that can specifically bind to the genomic DNA ormRNA of Campylobacter bacterial cdtA. The above-described methodsamplify a portion of genomic DNA or mRNA of C. coli cdtA using a primerpair that specifically binds to the genomic DNA or mRNA, and thus enabledetection of C. coli based on the presence or molecular weight of theamplified fragments. Specific examples of the above-described primersinclude the “primer pair comprising the sequences of SEQ ID NOs: 19 and20 (primers used in the Examples herein: Cc-CdtAU1 and Cc-CdtAR1)”. Theabove-described methods of the second embodiment enable simultaneousdetection of C. coli, and either or both of C. jejuni and C. fetus bysimultaneously using the above-described primer pair (a) in combinationwith “(b) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdtA, which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 21 and 22 (primers used in theExamples herein: Cj-CdtAU2 and Cj-CdtAR2) or mRNA region correspondingto the amplifiable genomic DNA region”, and/or “(c) a primer paircapable of amplifying a genomic DNA region of Campylobacter bacterialcdtA, which is amplified with a primer pair comprising the sequences ofSEQ ID NOs: 23 and 24 (primers used in the Examples herein: Cf-CdtAU1and Cf-CdtAR1) or mRNA region corresponding to the amplifiable genomicDNA region”, both of which comprise two polynucleotides that canspecifically bind to the genomic DNA or mRNA of Campylobacter bacterialcdtA.

The methods of the present invention comprise subsequent to theabove-described step of nucleic acid amplification reaction usingprimers specific to C. jejuni, C. fetus, or C. coli, the “step ofassessing the presence of Campylobacter bacteria based on the presenceor molecular weight of fragments amplified from genomic DNA or mRNA ofCampylobacter bacterial cdtA” or the “step of quantifying the amount offragments amplified from genomic DNA or mRNA of Campylobacter bacterialcdtA”. The amplified fragments of the present invention may be DNA orRNA.

The third embodiment of the methods of the present invention includesmethods for detecting Campylobacter bacteria in a test sample, whichcomprise the step of nucleic acid amplification reaction in the testsample using “(a) a primer pair capable of amplifying a genomic DNAregion of Campylobacter bacterial cdtC, which is amplified with a primerpair comprising the sequences of SEQ ID NOs: 25 and 26 (primers used inthe Examples herein: Cc-CdtCU1 and Cc-CdtCR1) or mRNA regioncorresponding to the amplifiable genomic DNA region”, which comprisestwo polynucleotides that can specifically bind to the genomic DNA ormRNA of Campylobacter bacterial cdtC. The above-described methodsamplify a portion of genomic DNA or mRNA of C. coli cdtC using a primerpair that specifically binds to the genomic DNA or mRNA, and thus enabledetection of C. coli based on the presence or molecular weight of theamplified fragments. The above-described methods of the third embodimentenable simultaneous detection of C. coli, and either or both of C.jejuni and C. fetus by simultaneously using the above-described primerpair (a) in combination with “(b) a primer pair capable of amplifying agenomic DNA region of Campylobacter bacterial cdtC, which is amplifiedwith a primer pair comprising the sequences of SEQ ID NOs: 27 and 28(primers used in the Examples herein: Cj-CdtCU1 and Cj-CdtCR2) or mRNAregion corresponding to the amplifiable genomic DNA region”, and/or “(c)a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdtC, which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 29 and 30 (primers used in theExamples herein: Cf-CdtCU2 and Cf-CdtCR1) or mRNA region correspondingto the amplifiable genomic DNA region”, both of which comprises twopolynucleotides that can specifically bind to the genomic DNA or mRNA ofCampylobacter bacterial cdtC.

The methods of the present invention comprise subsequent to theabove-described step of nucleic acid amplification reaction usingprimers specific to C. jejuni, C. fetus, and C. coli, the “step ofassessing the presence of Campylobacter bacteria based on the presenceor molecular weight of fragments amplified from genomic DNA or mRNA ofcdtC of the Campylobacter bacteria” or the “step of quantifying theamount of fragments amplified from genomic DNA or mRNA of cdtC of theCampylobacter bacteria”.

The fourth embodiment of the methods of the present invention includesmethods of detecting Campylobacter bacteria in a test sample, whichcomprise the step of nucleic acid amplification reaction in the testsample using any one or more of;

(a) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdt that is amplified by a primer paircomprising the sequences of SEQ ID NOs: 37 and 38, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

(b) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdt that is amplified by a primer paircomprising the sequences of SEQ ID NOs: 40 and 41, or an mRNA regioncorresponding to the amplifiable genomic DNA region; and

(c) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdt that is amplified by a primer paircomprising the sequences of SEQ ID NOs: 43 and 44, or an mRNA regioncorresponding to the amplifiable genomic DNA region;

each of which comprises two polynucleotides that can specifically bindto a genomic DNA or mRNA of cdt of the Campylobacter bacterium.

The above-described methods are methods for detecting bacteria in whicha portion of genomic DNA or mRNA of cdtC is amplified using primer pairsthat specifically bind to genomic DNA or mRNA of cdtC for each of C.jejuni, C. coli, and C. fetus, and the bacteria are detected based onthe presence or molecular weight of the amplified fragments.

For C. jejuni, the primers to be used in the above-described methodsfirst include the “primer pair comprising the sequences of SEQ ID NOs:37 and 38 (primers used in the Examples herein: Cj cdtRTU2 and CjcdtRTR2)”, but are not limited to these sequences. Any other sequencescan be used as long as the primer pairs comprise two polynucleotidesthat can specifically bind to genomic DNA or mRNA of C. jejuni cdtC,which is capable of amplifying a region amplified from genomic DNA of C.jejuni cdtC as a template using the “primer pair comprising thesequences of SEQ ID NOs: 37 and 38”, or the corresponding mRNA region.FIG. 12 shows the site where the “primer pair comprising the sequencesof SEQ ID NOs: 37 and 38” binds to the genomic DNA of cdtC for a totalof 11 C. jejuni strains. Furthermore, an amplification product is alsoobtained from a cdt gene (accession No. AY442300) defective C. jejunimutant by using the primer pair comprising the sequences of SEQ ID NOs:37 and 38.

As for C. coli, the primers to be used in the above-described methodsfirst include the “primer pair comprising the sequences of SEQ ID NOs:40 and 41 (primers used in the Examples herein: Cc cdtRTU5 and CccdtRTR5)”, but are not limited to the sequences. Any other sequences canbe used as long as the primer pairs comprise two polynucleotides thatcan specifically bind to genomic DNA or mRNA of C. coli cdtC as atemplate using the “primer pair comprising the sequences of SEQ ID NOs:40 and 41”, or amplify the corresponding mRNA region. FIG. 13 shows thesite where the “primer pair comprising the sequences of SEQ ID NOs: 40and 41” binds to the genomic DNA of cdtC for a total of 18 C. colistrains.

Furthermore, as for C. fetus, the primers to be used in theabove-described methods first include the “primer pair comprising thesequences of SEQ ID NOs: 43 and 44 (primers used in the Examples herein:Cf cdtRTU1 and Cf cdtRTR1)”, but are not limited to these sequences. Anyother sequences can be used as long as the primer pairs comprise twopolynucleotides that can specifically bind to genomic DNA or mRNA of C.fetus cdtC as a template using the “primer pair comprising the sequencesof SEQ ID NOs: 43 and 44, or amplify the corresponding mRNA region. FIG.15 shows the site where the “primer pair comprising the sequences of SEQID NOs: 43 and 44” binds to the genomic DNA of cdtC for a total of 12 C.fetus strains. Furthermore, with the primer pair comprising thesequences of SEQ ID NOs: 43 and 44, an amplification product for the cdtgene is also obtained from the C. fetus C90 strain isolated in Thailand.

In the methods of the present invention, it is possible to use thefollowing three types of primer pairs separately or combine and use themsimultaneously in single round nucleic acid amplification reaction:

“(a) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdt that is amplified by a primer paircomprising the sequences of SEQ ID NOs: 37 and 38, or an mRNA regioncorresponding to the amplifiable genomic DNA region”;

“(b) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdt that is amplified by a primer paircomprising the sequences of SEQ ID NOs: 40 and 41, or an mRNA regioncorresponding to the amplifiable genomic DNA region”; and

(c) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdt that is amplified by the primer paircomprising the sequences of SEQ ID NOs: 43 and 44, or an mRNA regioncorresponding to the amplifiable genomic DNA region. The presentinvention provides methods for detecting Campylobacter bacteria based onnucleic acid amplification methods, representatively including multiplexPCR using primers which are preferably used for amplifying differentnucleic acid regions and combinations thereof. There is no limitation onthe type of nucleic acid amplification method of the present inventionas long as it yields amplification products of interest. The PCR methodis an example of preferred nucleic acid amplification method in thepresent invention.

The methods of the present invention comprise subsequent to theabove-described step of nucleic acid amplification reaction usingprimers specific to C. jejuni, C. fetus, or C. coli, the “step ofassessing the presence of Campylobacter bacteria based on the presenceor molecular weight of fragments amplified from genomic DNA or mRNA ofCampylobacter bacterial cdtC” or the “step of quantifying the amount ofamplified fragments from genomic DNA or mRNA of Campylobacter bacterialcdtC”.

It is possible to use real-time PCR or such in the step of quantifyingthe amount of amplified fragments. Real-time PCR is a method formonitoring and analyzing the amount of fragments amplified by PCR inreal time. Real-time PCR is superior in rapidity and quantitativeperformance since there is no need for electrophoresis afteramplification of nucleic acid fragments. An example of quantitation ofbacterial cells by the real-time PCR method of the present invention isas follows. First, PCR is carried out using DNAs prepared by seriallydiluting a sample containing a known amount of bacterial cells as astandard, and then a standard curve is established by plotting theinitial DNA amount on the vertical axis and the cycle number (threshold;Ct value) that gives a predetermined amount of amplification productwithin the range of exponential amplification on the horizontal axis.Then, the Ct value of test samples is determined by PCR under the sameconditions. The bacterial cells are quantified by calculating the amountof DNA in the test samples from the standard curve.

Typically, real-time PCR is monitored using fluorescent reagents, andcarried out in a device integrating a thermal cycler and aspectrophotofluorometer. Fluorescence monitoring methods include knownmethods, such as use of intercalators and the TaqMan probe method, butare not limited thereto.

In the present invention, detection of nucleic acid fragments of thepresent invention amplified by real-time PCR can be achieved using, forexample, any one or more of:

“(i) the probe of SEQ ID NO: 39 (probe used in the Examples herein: CjRTP2) which can be used to detect nucleic acid fragments amplified witha primer pair comprising the sequences of SEQ ID NOs: 37 and 38”,

“(ii) the probe of SEQ ID NO: 42 (probe used in the Examples herein: CcRTP5) which can be used to detect nucleic acid fragments amplified witha primer pair comprising the sequences of SEQ ID NOs: 40 and 41”, and

“(iii) the probe of SEQ ID NO: 45 (probe used in the Examples herein: CfRTP1) which can be used to detect nucleic acid fragments amplified witha primer pair comprising the sequences of SEQ ID NOs: 43 and 44”.

It is preferred that these probes are appropriately conjugated with adetectable label. Such labeled components include fluorescentsubstances, radioisotopes, luminescent substances, enzymatically activesubstances, and magnetically-observable substances. The most preferablelabel in the present invention includes fluorescent labels.

When used in real-time PCR detection, probes are modified withfluorescent substance and quenching substance at the 5′ and 3′ ends,respectively. In the extension step of PCR, the probe that hybridizes tothe DNA region of the PCR template is degraded by the 5′-3′ exonucleaseactivity of DNA polymerase resulting in fluorescence emission, which isblocked by the quencher. Thus, the amplified fragment can be quantifiedbased on the fluorescence emission. In the present invention,fluorescent substances to be used for the modification include, forexample, FAM, TAMRA, and Orange560, but are not limited to theseexamples. Meanwhile, the quenching substances include, for example,Black Hole Quencher (BHQ), but are not limited thereto. As shown in theExamples, different amplified nucleic acid fragments can be detectedsimultaneously by attaching a different fluorescent label to each probe.

The methods of the present invention enable simple and rapid detectionof the presence of individual bacterial species of C. coli, C. jejuniand C. fetus in various biological samples from humans or animals (forexample, feces, rectal swab specimens, etc.) or food products. Themethods of the present invention can be conducted by usingpolynucleotide preparation methods (boil method or the like) known tothose skilled in the art to prepare polynucleotides from biologicalsamples, food products, or such, in which Campylobacter bacteria aresuspected to be present, and using the resulting polynucleotides as atest sample of the present invention.

<Kits>

The present invention provides kits to be used in the detection methodsof the present invention. The kits comprise manuals in addition to theprimer pairs of the present invention. The kits may further compriseother components, for example, fluorescent probes, intercalators, agentsfor preparing polynucleotides, and positive or negative primer pairs.

The first embodiment of the kits of the present invention includes kitscomprising at least one of:

“(a) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdtB which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 1 and 2, or an mRNA regioncorresponding to the amplifiable genomic DNA region” and “(b) a primerpair capable of amplifying a genomic DNA region of Campylobacterbacterial cdtB which is amplified with a primer pair comprising thesequences of SEQ ID NOs: 3 and 4, or an mRNA region corresponding to theamplifiable genomic DNA region”. The above-described primer pairs (a)and (b) allow amplification of regions specific (characteristic regions)to the genomic DNA or mRNA of C. jejuni and C. fetus cdtB, respectively.

As for C. jejuni, the above-described primers representatively include“primer pair comprising the sequences of SEQ ID NOs: 1 and 2 (primersused in the Examples herein: Cj-CdtBU5 and Cj-CdtBR6)”, but are notlimited to the sequences. Any other polynucleotides having a differentsequence can be used as long as they serve as a primer pair capable ofamplifying a region that is amplified from the genomic DNA or mRNA of C.jejuni cdtB as a template by using a “primer pair comprising thesequences of SEQ ID NOs: 1 and 2”, or the corresponding mRNA region.

Likewise, as for C. fetus, the above-described primers representativelyinclude “primer pair comprising the sequences of SEQ ID NOs: 3 and 4(primers used in the Examples herein: Cf-CdtBU6 and Cf-CdtBR3)”, but arenot limited to the sequences. Any other polynucleotides having adifferent sequence can be used as long as they serves as a primer paircapable of amplifying a region that is amplified from genomic DNA ormRNA of C. fetus cdtB as a template by using a “primer pair comprisingthe sequences of SEQ ID NOs: 3 and 4” or the corresponding mRNA region.

A “polynucleotide having a different sequence” that constitutes suchprimer pairs of the present invention is a polynucleotide of at least15, 20, or more nucleotides, which is complementary to the genomic DNAor mRNA of C. jejuni or C. fetus cdtB, for example, a polynucleotide of15 to 100 nucleotides, 20 to 100 nucleotides, 15 to 35 nucleotides, or20 to 35 nucleotides. Herein, “complementary strand” means one strandagainst the other in a double-stranded nucleic acid consisting of A:T (Uin the case of RNA) and G:C base pairs. Furthermore, “complementary”means that sequences are not necessarily fully complementary in a regionof at least 15 consecutive nucleotides but have at least 70%, preferablyat least 80%, more preferably 90%, still more preferably 95% or highernucleotide sequence homology. Algorithms for determining homologyinclude not only those described herein but also algorithms that areroutinely used by those skilled in the art for determining homology. Theabove-described “polynucleotides having a different sequence thatconstitute such primer pairs of the present invention” hybridize to cdtBgenomic DNA but not to DNAs encoding other polypeptides underhybridization conditions, preferably under stringent conditions.Furthermore, the primer pairs of the present invention do not hybridizeunder ordinary hybridization conditions, preferably under stringentconditions, to common regions of cdtB genomic DNA shared by variousCampylobacter bacteria. The above-described “polynucleotides having adifferent sequence that constitute such primer pairs of the presentinvention” are polynucleotides of at least 15 nucleotides, or 20 or morenucleotides, which comprise a nucleotide sequence with an addition,deletion, substitution, and/or insertion of one or more nucleotides (forexample, 1 to 10 nucleotides, or 1 to 5 nucleotides, preferably 1 to 4nucleotides, more preferably 1 to 3 nucleotides, and most preferably 1or 2 nucleotides) in the nucleotide sequence, for example, of any one ofSEQ ID NOs: 1, 2, 3, and 4.

The above-described “polynucleotides having a different sequence thatconstitute such primer pairs of the present invention” can beappropriately designed by those skilled in the art based on thepolynucleotide sequences of SEQ IDs shown above and/or known genomic DNAsequences of cdtB, and can be prepared by synthesis. Furthermore,whether polynucleotides prepared as described above are capable ofamplifying the same genomic DNA region as the original primer pairwithout mutation can be simply assessed by analyzing the amplifiedproducts resulting from nucleic acid amplification reaction using theprepared mutant primers.

The kits of the present invention may comprise primer pairs capable ofamplifying regions specific to genomic DNA or mRNA of C. coli cdtB, inaddition to the above-described primer pair (a) which is capable ofamplifying a region specific to genomic DNA or mRNA of C. jejuni cdtB,and the above-described primer pair capable of amplifying a regionspecific to genomic DNA or mRNA of C. fetus cdtB. Primer pairs that areindividually specific to each of the three species C. jejuni, C. fetus,and C. coli are all comprised in the kits of the present invention,allowing simultaneous detection of mixed infection with theabove-described Campylobacter bacteria by multiplex PCR or the like. Theabove-described primer pairs capable of amplifying regions specific togenomic DNA of C. coli cdtB include a “primer pair comprising thesequences of SEQ ID NOs: 5 and 6 (primers used in the Examples herein:Cc-CdtBU5 and Cc-CdtBR5)”, and other primer pairs capable of amplifyinga genomic DNA region of Campylobacter bacterial cdtB, which is amplifiedwith the “primer pair comprising the sequences of SEQ ID NOs: 5 and 6”,or mRNA region corresponding to the amplifiable genomic DNA region.

The second embodiment of the kits of the present invention include kitscomprising a primer pair of the present invention which is “(a) a primerpair capable of amplifying a genomic DNA region of Campylobacterbacterial cdtA, which can be amplified with a primer pair comprising thesequences of SEQ ID NOs: 19 and 20 (primers used in the Examples herein:Cc-CdtAU1 and Cc-CdtAR1), or mRNA region corresponding to theamplifiable genomic DNA region. The above-described primer pair (a)specifically binds to C. coli cdtA. The kits of the second embodimentmay comprise, in addition to primer pair (a), “(b) a primer pair capableof amplifying a genomic DNA region of Campylobacter bacterial cdtA,which is amplified with a primer pair comprising the sequences of SEQ IDNOs: 21 and 22 (primers used in the Examples herein: Cj-CdtAU2 andCj-CdtAR2), or an mRNA region corresponding to the amplifiable genomicDNA region”, and/or “(c) a primer pair capable of amplifying genomic DNAregion of Campylobacter bacterial cdtA, which is amplified with a primerpair comprising the sequences of SEQ ID NOs: 23 and 24 (primers used inthe Examples herein: Cf-CdtAU1 and Cf-CdtAR1), or an mRNA regioncorresponding to the amplifiable genomic DNA region”. It is thought thatthe kits comprising primer pairs (b) and/or (c) as well as primer pair(a) can simultaneously detect mixed infection with the above-describedCampylobacter bacteria when used in multiplex PCR or the like.

The third embodiment of the kits of the present invention include kitscomprising a primer pair of the present invention which is “(a) a primerpair capable of amplifying a genomic DNA region of Campylobacterbacterial cdtC, which is amplified with a primer pair comprising thesequences of SEQ ID NOs: 25 and 26 (primers used in the Examples herein:Cc-CdtCU1 and Cc-CdtCR1), or mRNA region corresponding to theamplifiable genomic DNA region. The above-described primer pair (a)specifically binds to C. coli cdtC. The kits of the third embodiment maycomprise, in addition to primer pair (a), “(b) a primer pair capable ofamplifying a genomic DNA region of Campylobacter bacterial cdtC, whichis amplified with a primer pair comprising the sequences of SEQ ID NOs:27 and 28 (primers used in the Examples herein: Cj-CdtCU1 andCj-CdtCR2), or mRNA region corresponding to the amplifiable genomic DNAregion”, and/or “(c) a primer pair capable of amplifying a genomic DNAregion of Campylobacter bacterial cdtC, which is amplified with a primerpair comprising the sequences of SEQ ID NOs: 29 and 30 (primers used inthe Examples herein: Cf-CdtCU2 and Cf-CdtCR1), or mRNA regioncorresponding to the amplifiable genomic DNA region”. It is thought thatthe kits comprising primer pairs (b) and/or (c) as well as primer pair(a) can simultaneously detect mixed infection with the above-describedCampylobacter bacteria when used in multiplex PCR or the like.

The fourth embodiment of the kits of the present invention includes kitscomprising as primer pair of the present invention at least one of:

“(a) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdt that is amplified by a primer paircomprising the sequences of SEQ ID NOs: 37 and 38, or an mRNA regioncorresponding to the amplifiable genomic DNA region (primers used in theExamples herein: Cj cdtRTU2 and Cj cdtRTR2)”;

“(b) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdt that is amplified by a primer paircomprising the sequences of SEQ ID NOs: 40 and 41, or an mRNA regioncorresponding to the amplifiable genomic DNA region (primers used in theExamples herein: Cc cdtRTU5 and Cc cdtRTR5)”, and

“(c) a primer pair capable of amplifying a genomic DNA region ofCampylobacter bacterial cdt that is amplified by a primer paircomprising the sequences of SEQ ID NOs: 43 and 44, or an mRNA regioncorresponding to the amplifiable genomic DNA region (primers used in theExamples: Cf cdtRTU1 and Cf cdtRTR1)”. Such kits comprising theabove-described primer pairs of (a), (b), and/or (c) are expected toenable simultaneous detection of mixed infection with Campylobacterbacteria by multiplex PCR or the like.

The above-described kits may further comprise any one or more of:

“(i) the probe of SEQ ID NO: 39 (probe used in the Examples herein: CjRTP2) which can be used to detect nucleic acid fragments amplified withthe primer pair comprising the sequences of SEQ ID NOs: 37 and 38”,

“(ii) the probe of SEQ ID NO: 42 (probe used in the Examples herein: CcRTP5) which can be used to detect nucleic acid fragments amplified withthe primer pair comprising the sequences of SEQ ID NOs: 40 and 41”, and

“(iii) the probe of SEQ ID NO: 45 (probe used in the Examples herein: CfRTP1) which can be used to detect nucleic acid fragments amplified withthe primer pair comprising the sequences of SEQ ID NOs: 43 and 44”.

The kits of the present invention may further comprise any one or moreof the above-described common primer pairs.

The type of nucleic acid amplification reaction using the kits of thepresent invention is not particularly limited, as long as it yieldsamplification products of interest. It is possible to select any type ofreaction from known reactions of nucleic acid amplification, forexample, the polymerase chain reaction (PCR) method (including RT-PCRmethod), ICAN method, LAMP method, SDA method, LCR method, and NASBAmethod. The preferred method is a PCR method.

The kits of the present invention may comprise not only theabove-described primer pairs and manual but also other materials. Othermaterials include, for example, positive primers, negative primers,agents for preparing polynucleotide, and fluorescently-labeled probes,but are not limited thereto. The positive primers can be appropriatelydesigned and prepared by those skilled in the art based on knownsequences of Campylobacter bacteria. Such known sequences ofCampylobacter bacteria are readily available in databases. For example,the 16S rRNA sequences of the C. jejuni ATCC 33560 strain, C. coli ATCC33559 strain, and C. fetus ATCC 27374 strain are available under theaccession NOs: M59298 (SEQ ID NO: 34), M59073 (SEQ ID NO: 35), andM65012 (SEQ ID NO: 36), respectively.

All prior-art documents cited herein are incorporated herein byreference.

EXAMPLES

Hereinbelow, the present invention is specifically described withreference to the Examples; however, it should not be construed as beinglimited thereto.

[1. Materials and Methods] (1-1 Bacterial Strains)

Bacteria used herein were: Campylobacter bacteria from ATCC, patients,and animals, cdt gene-positive non-Campylobacter bacteria, and otherbacteria responsible for enteric infection (Table 1). The C. jejuniCol-008 strain, C. coli Col-243 strain, C. fetus (Col-187 strain) wereused as positive control while the E. coli C600 strain was used asnegative control in PCR.

TABLE 1 (Campylobacter bacteria, Cdt-positive non-Campylobacterbacteria, and other bacteria responsible for enteric infection used toassess the multiplex PCR targeting the cdtB gene) Species Strain Origincdt gene C. jejuni Co1-008 Clinical + C. jejuni Co1-119 Clinical + C.jejuni Co1-126 Clinical + C. jejuni Co2-037 Clinical + C. jejuni Co2-127Clinical + C. jejuni Co2-128 Clinical + C. jejuni Co2-130 Clinical + C.jejuni Co2-132 Clinical + C. jejuni Co2-146 Clinical + C. jejuni Co2-150Clinical + C. jejuni Co2-193 Clinical + C. jejuni Co2-200 Clinical + C.jejuni Co2-214 Clinical + C. jejuni Co2-217 Clinical + C. jejuni Co3-007Clinical + C. jejuni Co3-008 Clinical + C. jejuni Co3-011 Clinical + C.jejuni Co3-012 Clinical + C. jejuni Co3-024 Clinical + C. jejuni Co3-036Clinical + C. jejuni Co3-072 Clinical + C. jejuni Co3-078 Clinical + C.jejuni Co3-082 Clinical + C. jejuni 81-176 Clinical + C. jejuni B01Bovine + C. jejuni B86 Bovine + C. jejuni 10114a Bovine + C. jejuni10114c Bovine + C. jejuni 8214c Bovine + C. jejuni 8215a Bovine + C.jejuni 8414c Bovine + C. jejuni 9914b Bovine + C. jejuni 33560 ATCC + C.jejuni 43432 ATCC + C. coli Co1-017 Clinical + C. coli Co1-071Clinical + C. coli Co1-106 clinical + C. coli Co1-124 clinical + C. coliCo1-192 clinical + C. coli Co1-130 clinical + C. coli Co1-194 clinical +C. coli Co1-245 clinical + C. coli Co1-247 clinical + C. coli Co2-060clinical + C. coli Co2-082 clinical + C. coli Co2-147 clinical + C. coliCo2-173 clinical + C. coli Co2-215 clinical + C. coli Co2-218 clinical +C. coli Co1-243 clinical + C. coli Co3-134 clinical + C. coli 33559ATCC + C. coli 43478 ATCC + C. fetus Co1-099 clinical + C. fetus Co1-187clinical + C. fetus 7914c Bovine + C. fetus 7915a Bovine + C. fetus7915b Bovine + C. fetus 8013a Bovine + C. fetus 8013b Bovine + C. fetus8013c Bovine + C. fetus 8614c Bovine + C. fetus 8813a Bovine + C. fetus8813c Bovine + C. fetus 9512a Bovine + C. fetus 9813a Bovine + C. fetus2-1 Bovine + C. fetus 3-1 Bovine + C. fetus 7 Bovine + C. fetus 23-1Bovine + C. fetus 86c Bovine + C. fetus 27374 ATCC + C. fetus 19438ATCC + C. lari 43675 ATCC + C. hyointestinalis 35217 ATCC + C.upsaliensis 43954 ATCC + C. heiveticus 51209 ATCC + H. hepaticus 51449ATCC + H. ducreyi 700724 ATCC + A. actinomycetemcomitans S01 Clinical +S. dysenteriae 155 AQ Clinical + S. dysenteriae SD-102 Clinical + S.dysenteriae 153 AQ Clinical + S. dysenteriae SD-104 Clinical + S.dysenteriae SD-107 Clinical + S. dysenteriae SD-112 Clinical + S. sonnei7 AQ Clinical + E. coli (cdtI) VS-1 Clinical + E. coli (cdtI) NT3363Clinical + E. coli (cdtI) GB1371 Clinical + E. coli (cdtI) P3 Clinical +E. coli (cdtI) b52 Animal + E. coli (cdtII) P101 Clinical + E. coli(cdtII) S9 Swine + E. coli (cdtIII) S45 Swine + E. coli (cdtIII) P183Clinical + E. coli (cdtIII) P194 Clinical + E. coli (cdtIII) B10Bovine + E. coli (cdtIII) b1 Bovine + E. coli (cdtIV) AQ25179 Clinical +E. coli (cdtIV) AQ13328 Clinical + E. coli (cdtIV) P132 Clinical + E.coli (cdtIV) P140 Clinical + E. coli (cdtIV) b95 Bivine + E. coli (cdtV)AQ11333 Clinical + E. coli (cdtV) P150 Clinical + E. coli (cdtV) P336Clinical + E. coli (cdtV) b1 Bovine + E. coli (cdtV) B102 Bovine +Salmonella spp. ST4, 311 Clinical − Salmonella spp. ST1, 312 Clinical −Salmonella spp. ST3, 307 Clinical − Salmonella spp. TM101 Clinical −Salmonella spp. TM103 Clinical − Salmonella spp. TM104 Clinical −Salmonella spp. TM105 Clinical − Salmonella spp. TM106 Clinical −Salmonella spp. TM107 Clinical − Salmonella spp. TM109 Clinical −Salmonella spp. TM110 Clinical − Salmonella spp. TM111 Clinical −Salmonella spp. TM112 Clinical − Salmonella spp. TM113 Clinical −Salmonella spp. TM114 Clinical − Salmonella spp. TM116 Clinical −Salmonella spp. TM117 Clinical − Salmonella spp. TM118 Clinical −Salmonella spp. TM119 Clinical − Salmonella spp. TM121 Clinical −Salmonella spp. TM122 Clinical − Salmonella spp. TM123 Clinical −Salmonella spp. TM125 Clinical − Salmonella spp. TM126 Clinical −Salmonella spp. TM127 Clinical − Salmonella spp. TM128 Clinical −Salmonella spp. TM129 Clinical − Salmonella spp. TM130 Clinical −Salmonella spp. TM132 Clinical − Salmonella spp. TM134 Clinical −Salmonella spp. TM135 Clinical − Salmonella spp. TM136 Clinical −Salmonella spp. TM137 Clinical − V. cholerae (O1) N16961 Clinical − V.cholerae (O1) 569B Clinical − V. cholerae (O1) VC406 Clinical − V.cholerae (non-O1/non-O139) C-l Clinical − V. cholerae (non-O1/non-O139)C-2 Clinical − V. cholerae (non-O1/non-O139) C-3 Clinical − V. cholerae(non-O1/non-O139) C-4 Clinical − V. cholerae (non-O1/non-O139) C-5Clinical − V. cholerae (non-O1/non-O139) C-6 Clinical − V. cholerae(non-O1/non-O139) C-7 Clinical − V. cholerae (non-O1/non-O139) C-8Clinical − V. cholerae (non-O1/non-O139) C-9 Clinical − V. cholerae(non-O1/non-O139) C-10 Clinical − V. cholerae (O1) A5 Clinical − V.cholerae (O1) A10 Clinical − V. cholerae (O1) A15 Clinical − V. cholerae(O1) B5 Clinical − V. cholerae (O1) B10 Clinical − V. cholerae (O1) C5Clinical − V. cholerae (O1) C9 Clinical − V. cholerae (O1) D5 Clinical −V. cholerae (O1) E5 Clinical − V. cholerae (O1) E10 Clinical − V.parahaemolyticus VP1 Shrimp − V. parahaemolyticus VP2 Shrimp − V.parahaemolyticus VP3 Shrimp − V. parahaemolyticus VP4 Shrimp − V.parahaemolyticus VP5 Shrimp − V. parahaemolyticus VP6 Shrimp − V.parahaemolyticus VP7 Shrimp − V. parahaemolyticus VP8 Shrimp − V.parahaemolyticus VP9 Shrimp − V. parahaemolyticus VP10 Shrimp − V.parahaemolyticus VP11 Shrimp − V. parahaemolyticus VP12 Shrimp − V.parahaemolyticus VP13 Shrimp − V. parahaemolyticus VP14 Shrimp − V.parahaemolyticus VP15 Shrimp − V. parahaemolyticus VP16 Shrimp − V.parahaemolyticus VP18 Shrimp − V. parahaemolyticus VP19 Shrimp − V.parahaemolyticus VP20 Shrimp − V. parahaemolyticus VP21 Shrimp − V.parahaemolyticus VP22 Shrimp − V. parahaemolyticus VP23 Shrimp − V.parahaemolyticus VP24 Shrimp − V. parahaemolyticus VP25 Shrimp − V.parahaemolyticus VP26 Shrimp − V. parahaemolyticus VP28 Shrimp − V.parahaemolyticus VP30 Shrimp − V. parahaemolyticus VP31 Shrimp − Y.enterocolitica Ye09 Clinical − S. dysenteriae SD1 Clinical − S.dysenteriae SD2 Clinical − S. dysenteriae SD3 Clinical − S. dysenteriaeSD5 Clinical − S. dysenteriae H14 174 Clinical − S. dysenteriae BCM519Clinical − S. dysenteriae HU29 Clinical − S. sonnei SS2 Clinical − S.sonnei SS3 Clinical − S. flexineri SF3 Clinical − S. flexineri SF4Clinical − S. flexineri SF5 Clinical − S. flexineri SF6 Clinical − S.flexineri SF7 Clinical − (1-2 Media, culture conditions, reagents, andenzymes)

Campylobacter bacteria, E. coli, and Shigella bacteria were cultured bythe following procedures. Campylobacter bacteria were cultured usinghorse blood agar plates containing CM271 BLOOD AGAR BASE No. 2 (Oxoid,Basingstoke, UK) [7.5 g of Proteose peptone, 1.25 g of Liver digest, 2.5g of Yeast extract, 2.5 g of NaCl, 6.0 g of Agar/500 ml of distilledwater (DW), pH 7.4±0.2 at 25° C.] supplemented with 5% steriledefibrinated horse blood (Nippon Bio-Supp. Center, Tokyo), and a mediumcontaining Campylobacter selective supplement (Skirrow) (OXOID) (5 mg ofVancomycin, 2.5 mg of Trimethoprim Lactate, 1,250 i.u. of PolymyxinB/500 ml) (hereinafter abbreviated as “Skirrow medium”). Campylobacterbacteria were cultured at 37° C. for two to four days under amicroaerophilic condition (10% CO₂, 5% O₂, and 85% N₂) using LOWTEMPERATURE O₂/CO₂ INCUBATER MODEL-9200 (WAKENYAKU CO, LTD., Tokyo). E.coli was cultured at 37° C. for 16 to 20 hours in liquid LB-Lenox medium(Difco Laboratories, Detroit, Mich., USA) (5.0 g of Bacto tryptone, 2.5g of Bacto yeast extract, 2.5 g of NaCl/500 ml of DW) or LB-Lenox agarplates (Difco Laboratories) (5.0 g of Bacto tryptone, 2.5 g of Bactoyeast extract, 2.5 g of NaCl, 7.5 g of Agar/500 ml of DW).

Helicobacter hepaticas (H. hepaticas) was cultured at 37° C. for 12 daysunder a microaerophilic condition (10% CO₂, 5% O₂, and 85% N₂) usingsheep blood agar plates containing Brucella Agar (Becton Dickinson,Franklin Lakes, N.J., USA) (5 g of Proteose peptone, 5 g of Pancreaticdigest of casein, 0.5 g of Dextrose, 1 g of Yeast extract, 2.5 g ofNaCl, 6.0 g of Agar/500 ml of DW, pH 7.4±0.2 at 25° C.) supplementedwith sterile defibrinated sheep blood (Nippon Bio-Supp. Center) at afinal concentration of 5%.

Haemophilus ducreyi (H. ducreyi) was cultured at 37° C. for seven daysunder a microaerophilic condition (10% CO₂, 5% O₂, and 85% N₂) using amedium prepared by the following procedure. Solution A [25 g of Heartinfusion broth (Difco Laboratories), 15 g of Agar/500 ml of DW, pH7.4±0.2 at 25° C.] and Solution B [10 g of Hemoglobin (BectonDickinson)/500 ml of DW] were sterilized by autoclaving at 121° C. for15 minutes, while Solution C [100 ml of Fetal bovine serum (Invitrogen),10 ml of IsoVitaleX (Becton Dickinson)] was filtrated. Solutions A, B,and C were mixed together to prepare the medium.

Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) wascultured at 37° C. for two days under an atmosphere of 90% air and 10%CO₂ in Trypticace soy agar (Becton Dickinson) (2.5 g of Papaic digest ofsoybean meal, 7.5 g Pancreatic digest of casein, 2.5 g of NaCl, 7.5 g ofAgar/500 ml of DW, pH 7.3±0.2 at 25° C.) containing 0.6% Yeast extract(Difco Laboratories).

Salmonella (Salmonella spp.) was cultured while shaking at 37° C. for 16to 20 hours in Trypticace soy broth (Becton Dickinson) (1.5 g of Papaicdigest of soybean meal, 8.5 g of Pancreatic digest of casein, 2.5 g ofNaCl, 1.25 g of K₂HPO₄, 1.25 g of Dextrose/500 ml of DW, pH 7.3±0.2 at25° C.).

Meanwhile, Yersinia enterocolitica was cultured while shaking at 30° C.for two days in Trypticace soy broth (Becton Dickinson).

Vibrio chorelae was cultured in LB-Lenox Broth (Difco Laboratories) at37° C. for 24 hours.

Vibrio parahaemolyticus (V. parahaemolyticus) was cultured at 37° C. for24 hours in alkaline peptone broth “Nissui” (Nissui, Tokyo) (5 g ofPeptone, 5 g of NaCl/500 ml of DW, pH 8.8±0.2 at 25° C.) containing 3%NaCl.

(1-3 PCR, Agarose Gel Electrophoresis, and Nucleotide Sequence Analysis)

Template DNAs for PCR were prepared by the boil method. Specifically,colonies were scraped from plates, and added to 200 μl of TE. Thesuspension was heated for ten minutes, and centrifuged at 12,800×g forten minutes (Himac CT13R, HITACHI; hereinafter the same centrifuge wasused unless otherwise specified). The resulting supernatants were usedas template DNA for PCR.

All PCR experiments were carried out using GeneAmp PCR System 2400(PerkinElmer; Wellesley, Mass., USA) or GeneAmp PCR System 9700(PerkinElmer). PCR primers and conditions used are listed in Table 2.Specifically, 5 μl each of degenerated PCR primers GNW and LPF-D (10μmol/μl), 40 ng of prepared genomic DNA, 4 μl of 2.5 mM dNTP, 5 μl of10× Ex Taq Buffer, and 0.25 μl of Takara Ex Taq (5 U/μl) were mixedtogether, and sterile DW was added to become 50 μl. The prepared mixturewas used in PCR. The PCR products were electrophoresed in 1% agarosegel. Agarose gel electrophoresis was carried out using a MUPID (ADVANCE,Tokyo) at 100 V in 1×TAE Buffer [40 mM Tris-acetate (pH 8.5), 1 mMEDTA]. After electrophoresis, the gel was stained with 1.0 μg/mlethidium bromide (Sigma) for 15 minutes. After destaining with DW, thePCR products were photographed under ultraviolet light (260 nm) usingGel Documentation System Gel Doc 2000 (Bio-Rad; Hercules, Calif., USA).

Nucleotide sequence analysis was conducted by the following procedure.100 ng of plasmid DNA was combined with 1 μl (3.2 μmol) each of thenucleotide sequencing primers listed in Table 4, 4 μl of Big Dyeterminator, and 2 μl of 5× sequence buffer. The volume was adjusted to20 μl with DW. PCR was carried out at 96° C. for five minutes, followedby 25 cycles of 96° C. for 30 seconds, 50° C. for 15 seconds, and 60° C.for four minutes. The PCR products were purified with CENTRI SPIN 20Spin Columns (Princeton Separations, Adelphia, N.J., USA), dried underreduced pressure using TOMY CENTRIFUGAL CONCENTRATOR CC-105 (TOMY SEIKOCO. LTD., Tokyo), and then dissolved in 20 ml of Template SuppressionReagent (Applied Biosystems, Foster City, Calif., USA). After boilingfor three minutes, the samples were rapidly cooled on ice. Thenucleotide sequences were determined using ABI PRISM 310 GeneticAnalyzer (Applied Biosystems). The resulting nucleotide sequences wereanalyzed with DNASIS (HitachiSoft, Tokyo) and Lasergene software(DNAstar, WI, USA). Furthermore, homology search was carried out usingBLAST (DDBJ, http://www.ddbj.nig.ac.jp/search/blast-j.html).

TABLE 2 (Multiplex PCR primers for the cdtA, cdtB, and cdtC genes of C.jejuni, C. coli, and C. fetus, common primer for the cdtB gene, and PCRconditions) PCR condition Name Sequence (5′-3′) Target DenaturingAnnealing Extention Amplicion (bp) C-CdtBcom1 ACTTGGAATTTGCAAGGCCj-/Cc-/Cf-cdtB 94° C., 30 s 50° C., 30 s 77° C., 30 s 717/723/712 (SEQID NO: 7) C-CdtBcom2 TCTAAAATTTACHGGAAAATG (SEQ ID NO: 8) Cj-CdtAU2AGGACTTGAACCTACTTTTC CjcdtA 94° C., 30 s 55° C., 30 s 72° C., 30 s 631(SEQ ID NO: 21) Cj-CdtAR2 AGGTGGAGTAGTTAAAAACC (SEQ ID NO: 22) Cj-CdtBU5ATCTTTTAACCTTGCTTTTGC CjcdtB 94° C., 30 s 56° C., 30 s 72° C., 30 s 714(SEQ ID NO: 1) Cj-CdtBR6 GCAAGCATTAAAATCGCAGC (SEQ ID NO: 2) Cj-CdtCU1TTTAGCCTTTGCAACTCCTA CjcdtC 94° C., 30 s 55° C., 30 s 72° C., 30 s 524(SEQ ID NO: 27) Cj-CdtCR2 AAGGGGTAGCAGCTGTTAA (SEQ ID NO: 28) Cc-CdtAU1ATTGCCAAGGCTAAAATCTC CccdtA 94° C., 30 s 55° C., 30 s 72° C., 30 s 329(SEQ ID NO: 19) Cc-CdtAR1 GATAAAGTCTCCAAAACTGC (SEQ ID NO: 20) Cc-CdtBU5TTTAATGTATTATTTGCCGC CccdtB 94° C., 30 s 56° C., 30 s 72° C., 30 s 413(SEQ ID NO: 5) Cc-CdtBR5 TCATTGCCTATGCGTATG (SEQ ID NO: 6) Cc-CdtCU1TAGGGATATGCACGCAAAAG CccdtC 94° C., 30 s 55° C., 30 s 72° C., 30 s 313(SEQ ID NO: 25) Cc-CdtCR1 GCTTAATACAGTTACGATAG (SEQ ID NO: 26) Cf-CdtAU1AACGACAAATGTAAGCACTC CfcdtA 94° C., 30 s 55° C., 30 s 72° C., 30 s 487(SEQ ID NO: 23) Cf-CdtAR1 TATTATGCAAGTCGTGCGA (SEQ ID NO: 24) Cf-CdtBU6GGCTTTGCAAAACCAGAAG CfcdtB 94° C., 30 s 56° C., 30 s 72° C., 30 s 553(SEQ ID NO: 3) Cf-CdtBR3 CAAGAGTTCCTCTTAAACTC (SEQ ID NO: 4) Cf-CdtCU2AAGCATAAGTTTTGCAAACG CfcdtC 94° C., 30 s 55° C., 30 s 72° C., 30 s 397(SEQ ID NO: 29) Cf-CdtCR1 GTTTGGATTTCAAATGTTCC (SEQ ID NO: 30) H: A, C,or T Cj, Co, and Cf are abbreviations for C. jejuni, C. coli, and C.fetus, respectively.

Example 1 Multiplex PCR for cdt Genes

For positive control, bacterial species-specific fragments of about 630bp, 330 bp, and 490 by were amplified by multiplex PCR for the cdtA genein C. jejuni, C. coli, and C. fetus, respectively. As in the case ofpositive control, bacterial species-specific fragments of about 710 bp,410 bp, and 550 b were amplified by multiplex PCR for the cdtB gene inC. jejuni, C. coli, and C. fetus, respectively. Furthermore, as in thecase of positive control, bacterial species-specific fragments of about500 bp, 300 bp, and 400 by were amplified by multiplex PCR for the cdtCgene in C. jejuni, C. coli, and C. fetus, respectively (FIG. 1).Meanwhile, none of the cdtA, cdtB, and cdtC genes was amplified in othercdt gene-positive Campylobacter bacteria such as C. hyointestinalis C.lari, C. upsaliensis, and C. helveticus; H. hepaticus, Haemophilusducreyi (H. ducreyi), A. actinomycetemcomitans, and Shigella spp.; andE. coli having five different types of cdt genes (I, II, III, IV, andV), while the cdt genes of the three bacterial species C. jejuni, C.coli, and C. fetus were specifically amplified (FIG. 1 and Table 3).Likewise, no amplified band was detected in other representativebacterial species responsible for enteric infection, includingSalmonella spp., Yersinia enterocolitica, and Vibrio spp. (Table 3).

TABLE 3 (Results of multiplex PCR and PCR by common primers in cdtgene-positive bacteria and other bacteria responsible for entericinfection isolated from patients and animals) C. jejuni C. coli C. fetusCommon Multiplex PCR Multiplex PCR Multiplex PCR PCR Species Origin (n*)cdtA cdtB cdtC cdtA cdtB cdtC cdtA cdtB cdtC cdtB C. jejuni Clinical(24) 24  24  24  — — — — — — 24 C. jejuni Animal (8) 8 8 8 — — — — — — 8C. jejuni ATCC33560 (1) 1 1 1 — — — — — — 1 C. jejuni ATCC43432 (1) 1 11 — — — — — — 1 C. coli Clinical (17) — — — 17  17  17  — — — 17 C. coliATCC33559 (1) — — — 1 1 1 — — — 1 C. coli ATCC43478 (1) — — — 1 1 1 — —— 1 C. fetus Clinical (2) — — — — — — 2 2 2 2 C. fetus Animal (16) — — —— — — 16  16  6 16 C. fetus subsp. fetus ATCC27374 (1) — — — — — — 1 1 11 C. fetus subsp. venerealis ATCC19438 (1) — — — — — — 1 1 1 1 C. lariATCC43675 (1) — — — — — — — — — — C. hyointestinalis ATCC35217 (1) — — —— — — — — — — C. helveticus ATCC51209 (1) — — — — — — — — — — C.upsaliensis ATCC43954 (1) — — — — — — — — — — H. hepticus ATCC51209 (1)— — — — — — — — — — H. ducreyi ATCC700724 (1) — — — — — — — — — — A.actinomycetemcomitans Clinical (1) — — — — — — — — — — Shigella spp.Clinical (21) — — — — — — — — — — E. coli (cdtI) Clinical and — — — — —— — — — — Aminal (5) E. coli (cdtII) Clinical and — — — — — — — — — —Aminal (2) E. coli (cdtIII) Clinical and — — — — — — — — — — Aminal (5)E. coli (cdtIV) Clinical and — — — — — — — — — — Aminal (5) E. coli(cdtV) Clinical and — — — — — — — — — — Aminal (5) Salmonella spp.Clinical (33) — — — — — — — — — — Y. enterocolitica Clinical (1) — — — —— — — — — — V. cholerae Clinical (23) — — — — — — — — — — V.parahaemolyticus Shrinp (28) — — — — — — — — — — Asterisk (*) representsthe number of strains examined.

Example 2 Detection of Campylobacter Bacteria Using Common Primers forthe cdtB Gene

Common primers targeting the cdtB gene, which has the highest homologybetween the bacterial species, were designed and used to assess whetherCampylobacter bacteria can be detected by a single round PCR targetingthe cdt gene. PCR using the common primers yielded bands of about 720 bythat are specific to the cdtB genes of C. jejuni, C. coli, and C. fetus.Furthermore, a fragment of about 720 by was also amplified in otherCampylobacter bacteria, specifically C. hyointestinalis, C. lari, C.upsaliensis, and C. helveticus, in addition to C. jejuni, C. coli, andC. fetus (FIG. 2). The nucleotide sequences of the obtained PCR productsof Campylobacter bacteria other than C. jejuni, C. coli, and C. fetuswere determined, and the result showed that each of them has a genehighly homologous to the cdtB gene of C. jejuni. Meanwhile, no fragmentwas amplified by the common primers in non-Campylobacter cdtgene-positive bacteria and other bacteria responsible for entericinfection, and even in H. hepaticus which was found to have cdt geneswith the highest homology to those of Campylobacter bacteria (FIG. 2 andTable 3). The result described above suggests that the cdtB genes ofCampylobacter bacteria contain a region conserved across the bacterialspecies. Thus, at least seven species of Campylobacter bacteria weredetectable in a single round PCR targeting the cdtB gene ofCampylobacter bacteria.

Example 3 Simultaneous Detection of Different Campylobacter Bacteria byMultiplex PCR Targeting the cdtB Gene

Under the assumption of mixed infection, it was assessed whethermultiplex PCR targeting the cdtB gene can simultaneously detectdifferent bacterial species of Campylobacter. In the previous Example,the multiplex PCR targeting the cdtA, cdtB, and cdtC genes wasdemonstrated to be specific to each of them. Since the cdtB gene wasfound to be most highly conserved among these subunit genes, multiplexPCR targeting the cdtB gene was used in the subsequent experiments.

Multiplex PCR targeting the cdtB gene was carried out by mixing two orthree of the genomic DNA of C. jejuni, C. coli, and C. fetus. As shownin the result of FIG. 3, specific bands for each species could beamplified not only when C. jejuni, C. coli, and C. fetus were presentseparately but also when two or three of them are present in mixture.Accordingly, multiplex PCR targeting the cdtB gene is expected to beapplicable in tests for mixed infection.

Example 4 Detection Limit of Multiplex PCR Targeting the cdtB Gene of C.jejuni, C. coli, and C. fetus

The detection limit of multiplex PCR targeting the cdtB gene wasevaluated using C. jejuni, C. coli, and C. fetus. The result showed that10¹ colony forming units (cfu) of bacterial cells per PCR tube wererequired for detection of the amplified fragment specific to C. jejunior C. coli. Meanwhile, 10² cfu of bacterial cells per PCR tube wererequired for detection of the amplified fragment specific to C. fetus(FIG. 4).

The sensitivity and specificity were proven to be improved by theprimers of the present invention as compared to other primers designedpreviously (SEQ ID NOs: 11 to 16 in WO 2005/054472). The primer sets ofthe present invention were used in PCR with boiled templates of ninesamples that had non-specific amplification when a previously designedprimer set was used, and non-specific amplification was not observed inany of the samples. Furthermore, 116 fecal samples from healthy childrenwere subjected to PCR using the primer sets of the present invention.Very weak non-specific amplification was observed in only one sample.

Example 5 Design of Primers and Probes Used for Detection of C. jejuni,C. coli, and C. fetus by Real-Time PCR

Primers and probes for C. jejuni, C. coli, and C. fetus were preparedbased on a total of 11 strains including cdtB gene-deficient strains, atotal of 18 strains including cdt gene-mutant strains, and a total of 12strains including the cdt gene-mutant strains isolated in Thailand,respectively. The best combination was selected for each of the bacteriaspecies by comparing the respective cdt gene sequences to specify theregions that allow detection of all bacterial strains in each of thebacteria species and to evaluate the specificity.

(1) C. jejuni

Comparison of a mutant deficient form of the C. jejuni cdt gene(AY442300) to the 81-176 strain cdt gene revealed that the mutantdeficient form of the cdt gene is deficient in cdtA and the first halfof cdtB as well as most of the midportion of cdtB, in addition to manynucleotide substitutions within the cdtB gene (FIG. 11). Thus, thepresent inventors focused on a relatively highly conserved region of thecdtC gene, and designed real-time PCR primers and probe within thisregion.

To design real-time PCR primers and probe, several cdtC genes includingdeficient forms were compared, and the region with the least mutationswhich can be used for the real-time PCR primers and probe is searched.Then, PCR was carried out using each primer set to select the mostsuitable primer set (FIG. 12).

Nucleotide Sequencing Primers

Cj cdtRTU2: 5′ GCAAAATCTTGTCAAGATGATCTAAAAG 3′ (SEQ ID NO: 37) CjcdtRTR2: 5′ TCCAAAACTAAAGAACGAATTTGCA 3′ (SEQ ID NO: 38)

Detection Probe

Cj RTP2: (SEQ ID NO: 39) 5′ (FAM)-AAACTGTATTTTCTATAATGCCAACAACAACTTCAG-(BHQ-1) 3′

BHQ: Black Hole Quencher (fluorescence quenching dye)

(2) C. coli

To design the real-time PCR primers and probe, several cdt genes of C.coli were compared, and the region with the least mutations which can beused for the real-time PCR primers and probe was searched. Then, PCR wascarried out using each primer set to select the most suitable primer set(FIG. 13).

Nucleotide Sequencing Primers

Cc cdtRTU5: 5′ TTTAACCAATGGTGGCAATCAAT 3′ (SEQ ID NO: 40) Cc cdtRTR5:5′ ATTCTCCTAAACCAAAGCGATTTTC 3′ (SEQ ID NO: 41)

Detection Probe

Cc RTP5: (SEQ ID NO: 42) 5′ (TAMRA)-CATGAGCACTTTTCCTGACTCTAGTATCGCCA-(BHQ-2) 3′(3) C. fetus

The cdt gene of several C. fetus strains was sequenced, and the resultshowed that the cdt gene of the C. fetus C90 strain isolated in Thailandwas different from that of the strains in Japan. The gene sequences werecompared between the two to search for highly conserved portions (FIG.14).

To design the real-time PCR primers and probe, several cdt genes of C.fetus were compared, and the region with the least mutations which canbe used for the real-time PCR primers and probe was searched. Then, PCRwas carried out using each primer set to select the most suitable primerset (FIG. 15).

Nucleotide Sequencing Primers

Cf cdtRTU1: 5′ CTTTTCCTTTTGGATACGTGCAA 3′ (SEQ ID NO: 43) Cf cdtRTR1:5′ AAAAATCCGCTAGGAGCGATCTG 3′ (SEQ ID NO: 44)

Detection Probe

Cf RTP1: (SEQ ID NO: 45) (Orange560)-CAAGTAGCAGCCGACGTAAAAATGTGCCT-(BHQ-1) 3′

Example 6 Detection and Detection Limit of C. jejuni, C. coli, and C.fetus by Real-Time PCR

Templates were prepared by the following procedure: bacterialsuspensions were prepared for each species of C. jejuni (81-176 strain),C. coli (Col-243 strain), and C. fetus (Col-187 strain) at 1, 10, 10²,10³, 10⁴, or 10⁵ cfu/μl, and then treated by the boil method (boilingfor 10 min, followed by centrifugation to obtain supernatants).Specifically, each PCR tube contained 1 to 10⁵ bacterial cells.

Real-time PCR was carried out by the following procedure: 1 ml each ofthe prepared templates was combined with 10 μl of TaqMan Master Mix(Applied Biosystems), 2 μl each of the above-described nucleotidesequencing primers (900 nM), and 1 ml each of the detection probes (250nM). The total volume was adjusted to 20 ml with DW. PCR was carried outunder the following conditions: heating at 95° C. for five minutes,followed by 40 cycles of 95° C. for 15 seconds and 60° C. for 60seconds. The reaction mixtures were stored at 4° C. The detection wasachieved using the Applied Biosystems 7500 Fast Real-Time PCR system(Applied Biosystems).

In the case of C. jejuni, at the concentration of 10⁵ cfu/tube,fluorescence emission started to increase at the 22nd cycle; at theconcentration of 10³ cfu/tube, fluorescence emission started to increaseat the 30th cycle; at the concentration of 10² cfu/tube, there was nosignificant fluorescence emission. The detection limit was 10³ cfu/tube(FIG. 8).

In the case of C. coli, at the concentration of 10⁵ cfu/tube,fluorescence emission started to increase at the 20th cycle; at theconcentration of 10² cfu/tube, fluorescence emission started to increaseat the 34th cycle. The detection limit was 10² cfu/tube (FIG. 9).

In the case of C. fetus, at the concentration of 10⁵ cfu/tube,fluorescence emission started to increase at the 21st cycle; at theconcentration of 10² cfu/tube, fluorescence emission started to increaseat the 35th cycle. The detection limit was 10² cfu/tube (FIG. 10).

Preferable results were obtained for the three bacterial species. Thus,the present invention was expected to provide sufficient performance forpractical use.

Probes were each labeled with a fluorescent substance that has differentfluorescence wavelength, and used to detect different bacterial species:FAM for C. jejuni; TAMRA for C. coli; and Orange560 for C. fetus. Thus,multiplex real-time PCR can be used to detect three bacterial species ina tube by using the Applied Biosystems 7500 Fast Real-Time PCR systemwhich is capable of multi-wavelength fluorescence detection.

INDUSTRIAL APPLICABILITY

The present invention provides novel methods for detecting Campylobacterbacteria and kits to be used in the detection methods. The methods ofthe present invention enable simple, rapid tests as compared toconventional methods. In particular, the methods of the presentinvention were demonstrated to be capable of identifying bacteria at thebacterial species level by carrying out multiplex PCR in the presence ofdifferent bacterial species of Campylobacter. As described above,Campylobacter bacteria are important from the viewpoint of publichygiene because they cause food poisoning. As a matter of fact,different bacterial species are often present together in infectedpatients or in the case of food contamination. Since the methods of thepresent invention can simultaneously detect bacteria without isolationof each species, they enable simple, rapid, and instantaneousidentification of bacteria that cause food poisoning or the like. Themethods of the present invention are very useful not only clinically butalso in the process management of food production or such, factoryhygiene management, or the like.

1. A method for detecting a Campylobacter bacterium in a test sample,which comprises the step of nucleic acid amplification reaction in thetest sample using one or more of primer pairs that comprise twopolynucleotides that can specifically bind to genomic DNA or mRNA ofcdtB of the Campylobacter bacterium, wherein the primer pairs are: (a) aprimer pair capable of amplifying a genomic DNA region of cdtB of theCampylobacter bacterium which is amplified with a primer pair comprisingthe sequences of SEQ ID NOs: 1 and 2, or an mRNA region corresponding tothe amplifiable genomic DNA region; and (b) a primer pair capable ofamplifying a genomic DNA region of cdtB of the Campylobacter bacteriumwhich is amplified with a primer pair comprising the sequences of SEQ IDNOs: 3 and 4, or an mRNA region corresponding to the amplifiable genomicDNA region.
 2. The method of claim 1, wherein the nucleic acidamplification reaction is carried out using primer pairs (a) and (b);and (c) a primer pair capable of amplifying a genomic DNA region of cdtBof the Campylobacter bacterium which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 5 and 6, or an mRNA regioncorresponding to the amplifiable genomic DNA region.
 3. The method ofclaim 1 or 2, which comprises, before or after the step of nucleic acidamplification reaction, an additional nucleic acid amplificationreaction step using a common primer pair comprising two polynucleotidesthat can commonly bind to the genomic DNA or mRNA of any one of cdtA,cdtB, and cdtC of Campylobacter bacteria.
 4. The method of claim 3,wherein the common primer pair is any one of: a primer pair comprisingthe sequences of SEQ ID NOs: 7 and 8; a primer pair comprising thesequences of SEQ ID NOs: 9 and 10; a primer pair comprising acombination of two sequences selected from the four sequences of SEQ IDNOs: 11, 12, 13, and 14; a primer pair comprising the sequences of SEQID NOs: 15 and 16; and a primer pair comprising the sequences of SEQ IDNOs: 17 and
 18. 5. A kit for use in the method of claim 1, whichcomprises a manual and at least either: (a) a primer pair capable ofamplifying a genomic DNA region of cdtB of the Campylobacter bacteriumwhich is amplified with the primer pair comprising the sequences of SEQID NOs: 1 and 2, or an mRNA region corresponding to the amplifiablegenomic DNA region; or (b) a primer pair capable of amplifying a genomicDNA region of cdtB of the Campylobacter bacterium which is amplifiedwith the primer pair comprising the sequences of SEQ ID NOs: 3 and 4, oran mRNA region corresponding to the amplifiable genomic DNA region; eachof which comprises two polynucleotides that can specifically bind to thegenomic DNA or mRNA of cdtB of the Campylobacter bacterium.
 6. The kitof claim 5, which further comprises: (c) a primer pair capable ofamplifying a genomic DNA region of cdtB of the Campylobacter bacteriumwhich is amplified with the primer pair comprising the sequences of SEQID NOs: 5 and 6, or an mRNA region corresponding to the amplifiablegenomic DNA region.
 7. A method for detecting a Campylobacter bacteriumin a test sample, which comprises the step of nucleic acid amplificationreaction in the test sample using a primer pair comprising twopolynucleotides that can specifically bind to genomic DNA or mRNA ofcdtA of a Campylobacter bacterium, wherein the primer pair is: (a) aprimer pair capable of amplifying a genomic DNA region of cdtA of theCampylobacter bacterium which is amplified with a primer pair comprisingthe sequences of SEQ ID NOs: 19 and 20, or an mRNA region correspondingto the amplifiable genomic DNA region.
 8. The method of claim 7, whereinthe nucleic acid amplification reaction is carried out using primer pair(a), and (b) a primer pair capable of amplifying a genomic DNA region ofcdtA of the Campylobacter bacterium which is amplified with a primerpair comprising the sequences of SEQ ID NOs: 21 and 22, or an mRNAregion corresponding to the amplifiable genomic DNA region; and (c) aprimer pair capable of amplifying a genomic DNA region of cdtA of theCampylobacter bacterium which is amplified with a primer pair comprisingthe sequences of SEQ ID NOs: 23 and 24, or an mRNA region correspondingto the amplifiable genomic DNA region.
 9. A kit for use in the method ofclaim 7, which comprises a manual and a primer pair comprising twopolynucleotides that can specifically bind to the genomic DNA or mRNA ofcdtA of the Campylobacter bacterium, wherein the primer pair is: (a) aprimer pair capable of amplifying a genomic DNA region of cdtA of theCampylobacter bacterium which is amplified with the primer paircomprising the sequences of SEQ ID NOs: 19 and 20, or an mRNA regioncorresponding to the amplifiable genomic DNA region.
 10. The kit ofclaim 9, which further comprises: (b) a primer pair capable ofamplifying a genomic DNA region of cdtA of the Campylobacter bacteriumwhich is amplified with the primer pair comprising the sequences of SEQID NOs: 21 and 22, or an mRNA region corresponding to the amplifiablegenomic DNA region; and (c) a primer pair capable of amplifying agenomic DNA region of cdtA of the Campylobacter bacterium which isamplified with the primer pair comprising the sequences of SEQ ID NOs:23 and 24, or an mRNA region corresponding to the amplifiable genomicDNA region.
 11. A method for detecting a Campylobacter bacterium in atest sample, which comprises the step of nucleic acid amplificationreaction in the test sample using a primer pair comprising twopolynucleotides that can specifically bind to genomic DNA or mRNA ofcdtC of a Campylobacter bacterium, wherein the primer pair is: (a) aprimer pair capable of amplifying a genomic DNA region of cdtC of theCampylobacter bacterium which is amplified with a primer pair comprisingthe sequences of SEQ ID NOs: 25 and 26, or an mRNA region correspondingto the amplifiable genomic DNA region.
 12. The method of claim 11,wherein the nucleic acid amplification reaction is carried out usingprimer pair (a), and (b) a primer pair capable of amplifying a genomicDNA region of cdtC of the Campylobacter bacterium which is amplifiedwith a primer pair comprising the sequences of SEQ ID NOs: 27 and 28, oran mRNA region corresponding to the amplifiable genomic DNA region; and(c) a primer pair capable of amplifying a genomic DNA region of cdtC ofthe Campylobacter bacterium which is amplified with a primer paircomprising the sequences of SEQ ID NOs: 29 and 30, or an mRNA regioncorresponding to the amplifiable genomic DNA region.
 13. A kit for usein the method of claim 11, which comprises a manual and a primer paircomprising two polynucleotides that can specifically bind to the genomicDNA or mRNA of cdtC of the Campylobacter bacterium, wherein the primerpair is: (a) a primer pair capable of amplifying a genomic DNA region ofcdtC of the Campylobacter bacterium which is amplified with the primerpair comprising the sequences of SEQ ID NOs: 25 and 26, or an mRNAregion corresponding to the amplifiable genomic DNA region.
 14. The kitof claim 13, which further comprises: (b) a primer pair capable ofamplifying a genomic DNA region of cdtC of the Campylobacter bacteriumwhich is amplified with the primer pair comprising the sequences of SEQID NOs: 27 and 28, or an mRNA region corresponding to the amplifiablegenomic DNA region; and (c) a primer pair capable of amplifying agenomic DNA region of cdtC of the Campylobacter bacterium which isamplified with the primer pair comprising the sequences of SEQ ID NOs:29 and 30, or an mRNA region corresponding to the amplifiable genomicDNA region.
 15. A method for detecting a Campylobacter bacterium in atest sample, which comprises the step of nucleic acid amplificationreaction in a test sample using one or more of: (a) a primer paircapable of amplifying a genomic DNA region of cdtC of a Campylobacterbacterium that is amplified by a primer pair comprising the sequences ofSEQ ID NOs: 37 and 38, or an mRNA region corresponding to theamplifiable genomic DNA region; (b) a primer pair capable of amplifyinga genomic DNA region of cdtC of a Campylobacter bacterium that isamplified by a primer pair comprising the sequences of SEQ ID NOs: 40and 41, or an mRNA region corresponding to the amplifiable genomic DNAregion; and (c) a primer pair capable of amplifying a genomic DNA regionof cdtC of a Campylobacter bacterium that is amplified by a primer paircomprising the sequences of SEQ ID NOs: 43 and 44, or an mRNA regioncorresponding to the amplifiable genomic DNA region; each of whichcomprises two polynucleotides that can specifically bind to a genomicDNA or mRNA of cdt of the Campylobacter bacterium.
 16. The method ofclaim 15, wherein the nucleic acid amplification reaction is achieved byusing a quantitative PCR method or quantitative real-time PCR method.17. The method of claim 15 or 16, which further comprises any one ormore of: (i) the step of detecting the nucleic acid fragment amplifiedwith a primer pair comprising the sequences of SEQ ID NOs: 37 and 38 byusing the probe of SEQ ID NO: 39; (ii) the step of detecting the nucleicacid fragment amplified with a primer pair comprising the sequences ofSEQ ID NOs: 40 and 41 by using the probe of SEQ ID NO: 42; and (iii) thestep of detecting the nucleic acid fragment amplified with a primer paircomprising the sequences of SEQ ID NOs: 43 and 44 by using the probe ofSEQ ID NO:
 45. 18. A kit for use in the method of claim 15, whichcomprises a manual and at least one of: (a) a primer pair capable ofamplifying a genomic DNA region of cdt of a Campylobacter bacterium thatis amplified by the primer pair comprising the sequences of SEQ ID NOs:37 and 38, or an mRNA region corresponding to the amplifiable genomicDNA region; (b) a primer pair capable of amplifying a genomic DNA regionof cdt of a Campylobacter bacterium that is amplified by the primer paircomprising the sequences of SEQ ID NOs: 40 and 41, or an mRNA regioncorresponding to the amplifiable genomic DNA region; and (c) a primerpair capable of amplifying a genomic DNA region of cdt of aCampylobacter bacterium that is amplified by the primer pair comprisingthe sequences of SEQ ID NOs: 43 and 44, or an mRNA region correspondingto the amplifiable genomic DNA region; each of which comprises twopolynucleotides that can specifically bind to a genomic DNA or mRNA ofcdt of the Campylobacter bacterium.
 19. The kit of claim 18, whichfurther comprises at least one of the probes of SEQ ID NOs: 39, 42, and45.